Antibacterial and antibiotic-potentiation activities of the hydro-ethanolic extract and protoberberine alkaloids from the stem bark of Enantia chlorantha against multidrug-resistant bacteria expressing active efflux pumps.

ETHNOPHARMACOLOGICAL RELEVANCE: Enantia chlorantha is traditionally used to treat various ailments including rickettsia fever, cough and wounds, typhoid fever, infective hepatitis, jaundice, and urinary tract infections. AIM OF THE STUDY: To isolate the antibacterial constituents of the hydro-ethanolic extract of the stem bark of E. chlorantha (ECB) and to evaluate the antibacterial and antibiotic-modifying activities of ECB and its constituents against the multidrug-resistant (MDR) phenotypes. MATERIALS AND METHODS: Chromatographic methods were used to isolate the constituents of ECB and Spectroscopic methods were used to elucidate the chemical structures of the isolated compounds. The antibacterial activity of samples was determined by the broth microdilution method while spectrophotometric methods were used to evaluate the effects of ECB and its most active constituent on bacterial growth. Their effects on bacterial proton-ATPase pumps was assessed through the acidification of the bacterial culture medium. RESULTS: Six protoberberine alkaloids were isolated and identified as columbamine (1), pseudocolumbamine (2), jathrorrhizine (3), palmitine (4), 4,13-dihydroxy-3,9,10-trimethoxyprotoberberine (5), and 13-hydroxy-2,3,9,10-tetramethoxyprotoberberine (6). The crude extract (ECB) inhibited the growth of all the tested MDR bacteria, with the minimal inhibitory concentration (MIC) values below 100 µg/mL obtained against Escherichia coli ATCC 10536, AG 102, Enterobacter aerogenes EA 27, Klebsiella pneumoniae ATCC 11296 and KP 55, Providencia stuartii NEA 16, and Staphylococcus aureus MRSA3 and MRSA6. Compound 1 had the best antibacterial effects with MIC values ranging from 16 to 64 µg/mL. The efflux pump inhibitor (EPI), phenylalanine-arginine-ß naphthylamide (PAßN) significantly improved the activity of compounds 1-6. Compounds 1-3 significantly potentiated the antibacterial activity of antibiotics such norfloxacin (NOR), ciprofloxacin (CIP), and doxycycline (DOX) against the tested MDR bacteria. CONCLUSION: The crude extract (ECB) and its isolated compounds 1-6 are potential antibacterial products from Enantia chlorantha. They could be explored more to develop the antibacterial agents that could be used alone or in combination with antibiotics to overcome MDR phenotypes.

Cytotoxicity, acute and sub-chronic toxicities of the fruit extract of Tetrapleura tetraptera (Schumm. & Thonn.) Taub. (Fabaceae).

BACKGROUND: Tetrapleura tetraptera is a medicinal spice traditionally used to treat cancer, diabetes, and several other ailments. This study analyzed the cytotoxicity of the dichloromethane methanol extract of T. tetraptera fruits (TTF) and its constituents. The toxicity profile of the TTF extract was also evaluated in rats. METHODS: The Cytotoxicity of this extract was evaluated using the resazurin reduction assay (RRA). Acute and sub-chronic toxicity studies were performed according to the protocol described by the Organisation for Economic Cooperation, and Development (OECD). Hematological, serum, and urine biochemical parameters, as well as histological sections of the liver and kidney, were also evaluated based on standard methods. RESULTS: The TTF extract, compound 5, and the reference drug doxorubicin were active in all 9 tested cancer cell lines. The recorded IC50 ranged from 18.32 µM (against B16-F1 murine melanoma cells) to 36.18 µM (against SKMel-505 BRAF wildtype melanoma cells) for TTF, from 10.02 µM (towards MaMel-80a BRAF-V600E homozygous mutant melanoma cells) to 31.73 µM (against SKMel-28 BRAF-V600E homozygous mutant melanoma cells) for compound 5, and from 0.22 µM (against B16-F1 cells) to 9.39 µM (against SKMel-505 cells) for doxorubicin. The study of acute toxicity test showed that the lethal dose (LD50) of this extract was greater than 5000 mg/kg body weight. In the sub-chronic toxicity studies, variations were observed in some biochemical parameters, especially at higher doses. CONCLUSION: TTF and its most active compound (5) are found to be potential cytotoxic agents, meanwhile, TTF was safe when given a single oral dose of 5000 mg/kg. However, caution is necessary in case of prolonged oral administration due to potential alterations of renal function at high doses (> 1000 mg/kg).

Antibacterial and antibiotic-potentiation activity of the constituents from aerial part of Donella welwitshii (Sapotaceae) against multidrug resistant phenotypes.

BACKGROUND: The rise of multidrug-resistant (MDR) bacteria is a real public health problem worldwide and is responsible for the increase in hospital infections. Donella welwitschii is a liana or shrub belonging to the family Sapotaceae and traditionally used to cure coughs. OBJECTIVE: This study was conducted with the objective to validate the medicinal properties of this plant, the aerial part was studied for its phytochemical composition using column and PTLC chromatography and exploring its antibacterial and antibiotic-modifying activity as well as those of its phytochemicals. METHODS: The structures of the compounds were elucidated from their physical and spectroscopic data in conjunction with literature. The antibacterial activity of the isolated metabolites was performed toward a panel of MDR Gram negative and Gram-positive bacteria. The broth micro-dilution method was used to determine antibacterial activities, efflux pump effect using the efflux pump inhibitor (EPI) (phenylalanine-arginine-ß-naphthylamide (PAßN)), as well as the modulating activity of antibiotics. Monitoring the acidification of the bacterial growth medium was used to study the effects of the samples on the bacterial proton-ATPase pumps and cellular ATP production. RESULTS: Eleven compounds were isolated including pentacyclic triterpenes, C-glucosyl benzophenones. With a MIC value < 10 µg/mL, diospyric acid (7) significantly inhibited the growth of Escherichia coli AG102, Enterobacter aerogenes ATCC13048, Klebsiella pneumoniae KP55, Providencia stuartii NEA16 and Staphylococcus aureus MRSA3. 28-hydroxy-ß-amyrin (8) significantly impaired the growth of Enterobacter aerogenes EA27, Klebsiella pneumoniae ATCC11296 and Staphylococcus aureus MRSA6; and oleanolic acid (9) strongly impaired the growth of Escherichia coli AG 102, Enterobacter aerogenes EA27 and Providencia stuartii PS2636. Diospyric acid (7) and 28-hydroxy-ß-amyrin (8) induced perturbation of H+-ATPase pump and inhibition of the cellular ATP production. Moreover, at MIC/2 and MIC/4, compounds 7, 8, and 9 strongly improved the antibacterial activity of norfloxacin, ciprofloxacin and doxycycline with antibiotic-modulating factors ranging between 2 and 64. CONCLUSION: The overall results of the current work demonstrate that diospyric acid (7), 28-hydroxy-ß-amyrin (8) and oleanolic acid (9) are the major bioactive constituents of Donella welwitschia towards Gram-negative bacteria expressing MDR phenotypes.

Botanical from the Fruits Mesocarp of Raphia vinifera Displays Antiproliferative Activity and Is Harmless as Evidenced by Toxicological Assessments.

Raphia vinifera is widely used to treat several diseases including digestive disorders, dysentery, and genitourinary infections. In this study, the mineral contents, the cytotoxicity, and the toxicological effect of the crude CHCl3/MeOH extract (RVM) from the mesocarp of Raphia vinifera were evaluated. The mineral contents were evaluated using the method described by the Association of Official Analytical Chemists (AOAC). The cytotoxicity of both extract and chemical compounds from the plants was determined by a resazurin reduction assay (RRA). The toxicological studies were carried out using the experimental procedure of the Organization for Economic Cooperation and Development (OECD). After killing the rats, biochemical, histopathological, and hematological studies were performed. The result indicated that RVM is rich in zinc (6.52 mg/100 g of DM) and sodium (194.5 mg/100 g of DM). RVM had a cytotoxicity effect with IC50 values lower than 30 µg/mL in 18/18 cancer cell lines tested. These recorded IC50 values were between 12.35 µg/mL (toward CCRF-CEM leukemia cells) and 26.66 µg/mL (toward SKMel-505 BRAF wild-type melanoma cells). Raphvinin 4 displayed good cytotoxicity against MaMel-80aBRAF-V600E homozygous mutant with the IC50 of 10.42 µM. RVM was relatively nontoxic to rats, the median lethal dose (DL50) being above 5000 mg/kg body weight. However, during the oral administration period extending for 28 days, precautions should be taken due to the increase in urinary creatinine level and decrease in spleen weight in the male rats given the highest dose (1000 mg/kg) of extract. Conclusively, the extract of Raphia vinifera is weakly toxic in rats and could be further used in the development of anticancer phytomedicines.

Cytotoxic phytochemicals from the crude extract of Tetrapleura tetraptera fruits towards multi-factorial drug resistant cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Tetrapleura tetraptera is an African medicinal spice used in traditional medicine to treat several ailments including cancer. AIM OF THE STUDY: The present study was designed to evaluate the cytotoxicity of the dichloromethane-methanol (1:1) extract of the fruits of Tetrapleura tetraptera (TTF) and its constituents: (3R, 4S)-3,4-dimethyloxetan-2-one (1), luteolin (2), stigmasterol (4), 3-O-[6'-O-undecanoyl-ß-D-glucopyranosyl]stigmasterol (6), olean-12-en-3-ß-O-D-glucopyranoside (7), 3-O-ß-D-glucopyranosyl-(1 â†’ 6)-ß-D-glucopyranosylurs-12-en-28-oic acid (8), 3-O-ß-D-glucopyranosyl-(1 â†’ 3)-ß-D-glucopyranosyl-27-hydroxyolean-12-ene-28-oic acid (9), methyl-O-ß-D-glucopyranoside (10), ß-D-fructofuranosyl-(2 â†’ 1)-ß-D-glucopyranoside (11) towards a panel of cancer cell lines including MDR phenotypes. The cellular mode of induction of apoptosis by TTF and compound 7 was further investigated. MATERIALS AND METHODS: The resazurin reduction assay (RRA) was applied to determine the cytotoxicity of the studied samples. The cell cycle (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP; JC-1) and reactive oxygen species (ROS; H2DCFH-DA) were measured by flow cytometry. Column chromatography was used for the purification of TTF, whilst nuclear magnetic resonance (NMR) spectroscopic analysis was applied for structural elucidation. RESULTS: The botanical, TTF and the phytochemicals, 2, 7, 8 and 9 as well as doxorubicin exerted cytotoxicity against 9 cancer cell lines including drug-sensitive and drug resistant phenotypes. TTF, compound 7 and doxorubicin were the most active samples, and displayed IC50 values ranging from 10.27 µg/mL (in CCRF-CEM leukemia cells) to 23.61 µg/mL (against HCT116 p53-/- colon adenocarcinoma cells) for TTF, from 4.76 µM (against CCRF-CEM cells) to 12.92 µM (against HepG2 hepatocarcinoma cells) for compound 7, and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin. TTF induced apoptosis in CCRF-CEM cells through MMP alteration and increased ROS production while compound 7 induced apoptosis mediated by caspases activation, MMP alteration and increased ROS production. CONCLUSION: Tetrapleura tetraptera and some of its constituents, mostly compound 7 are good cytotoxic natural products that should be explored in depth to develop new drugs to fight cancers.

N-acetylglycoside of oleanolic acid (aridanin) displays promising cytotoxicity towards human and animal cancer cells, inducing apoptotic, ferroptotic and necroptotic cell death.

BACKGROUND: The discovery of novel phytochemicals represents a reasonable approach to fight malignancies, especially those which are resistant to standard chemotherapy. PURPOSE: We evaluated the cytotoxic potential of a naturally occurring N-acetylglycoside of oleanolic acid, aridanin, on 18 cancer cell lines, including sensitive and drug-resistant phenotypes mediated by P-glycoprotein, BCRP, p53 knockout, deletion-mutated EGFR, or BRAF mutations. Furthermore, metastasizing B16/F10 cells, HepG2 hepatocarcinoma and normal AML12 hepatocytes were investigated. The mechanisms of aridanin-induced cell death was further investigated. METHODS: The resazurin reduction assay (RRA) was applied to evaluate the cytotoxicity, autophagy, ferroptotic and necroptotic cell death. CCRF-CEM leukemia cells were used for all mechanistic studies. A caspase-Glo assay was applied to evaluate the caspase activities. Flow cytometry was applied for the analyses of cell cycle (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP; JC-1) and reactive oxygen species (ROS; H2DCFH-DA). RESULTS: Aridanin and doxorubicin (positive control) inhibited the proliferation of all cancer cell lines tested. The IC50 values for aridanin varied from 3.18 µM (CCRF-CEM cells) to 9.56 µM (HepG2 cells). Aridanin had considerably lower IC50 values than that of doxorubicin against multidrug-resistant CEM/ADR5000 cells and melanoma cell lines (MaMel-80a, Mel-2a, MV3, and SKMel-505). Aridanin induced apoptosis in CCRF-CEM cells through increase of ROS levels and MMP breakdown, and to a lesser extent via caspases activation. Aridanin also induced ferroptotic and necroptotic cell death. CONCLUSION: The present study opens good perpectives for the use of this phytochemical as an anticancer drug to combat multi-facorial resistance to established chemotherapeutics.

Cytotoxicity of a naturally occuring spirostanol saponin, progenin III, towards a broad range of cancer cell lines by induction of apoptosis, autophagy and necroptosis.

This study was aimed to investigate the cytotoxic potential of a natural compound, progenin III on a broad range of cancer cell lines, including various sensitive and drug-resistant phenotypes. The cytotoxicity, progenin III-induced autophagic, ferroptotic and necroptotic cell death were evaluated by the resazurin reduction assay (RRA). Spectrophotometric analysis of caspases activity was performed using caspase-Glo assay. Flow cytometry was applied for cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H2DCFH-DA). Progenin III and the reference molecule, doxorubicin exerted cytotoxic effects towards the 18 cancer cell lines tested including animal and human cell lines. The IC50 values obtained ranged from 1.59 µM (towards CCRF-CEM leukemia cells) to 31.61 µM (against the BRAF-V600E homozygous mutant SKMel-28 melanoma cells) for progenin III. Normal sensitivity was achieved with CEM/ADR5000 cells and HCT116p53-/- adenocarcinoma cells respectively compared to their sensitive congeners CCRF-CEM cells and HCT116 p53+/+ cells. Progenin III induced apoptosis in CCRF-CEM cells mediated by caspases 3/7 activation, MMP alteration and increase ROS production, and otherwise autophagy and necroptosis. Progenin III is a potential anticancer molecule that deserves further investigations to develop a novel drug to combat malignant diseases including refractory cancers.