An Efflux Pumps Inhibitor Significantly Improved the Antibacterial Activity of Botanicals from Plectranthus glandulosus towards MDR Phenotypes.

Bacterial multidrug resistance causes many therapeutic failures, making it more difficult to fight against bacterial diseases. This study aimed to investigate the antibacterial activity of extract, fractions, and phytochemicals from Plectranthus glandulosus (Lamiaceae) against multidrug-resistant (MDR) Gram-negative phenotypes expressing efflux pumps. The crude extract after extraction was subjected to column chromatography, and the structures of the isolated compounds were determined using spectrometric and spectroscopic techniques. Antibacterial assays of samples alone and in the presence of an efflux pump inhibitor (phenylalanine-arginine ß-naphthylamide, PAßN) were carried out using the broth microdilution method. The phytochemical study of P. glandulosus plant extract afforded seven major fractions (A-G) which lead to the isolation of seventeen known compounds. The ethanol extract of P. glandulosus was not active at up to 1024 µg/mL, whereas its fractions showed MICs varying from 32 to 512 µg/mL on the studied bacteria. Fraction C of P. glandulosus showed the lowest MIC (32 µg/mL) on E. coli ATCC8739 strain. Fraction D presented the highest activity spectrum by inhibiting the growth of 90% (9/10) of the studied bacteria. The presence of PAßN has improved the activity of extract and all fractions. Overall, the tested phytochemicals showed low activity against the studied bacteria. The overall results obtained in this study show that some fractions from P. glandulosus, mainly fractions C and D, should be investigated more for their possible use to fight against MDR bacteria.

Cytotoxicity of Crude Extract and Isolated Constituents of the Dichrostachys cinerea Bark towards Multifactorial Drug-Resistant Cancer Cells.

The effectiveness of anticancer chemotherapy is greatly impeded by the resistance of malignant cells to cytotoxic drugs. In this study, the cytotoxicity of the crude extract (DCB) and compounds isolated from the bark of Dichrostachys cinerea, namely, betulinic acid (1), glyceryl-1-hexacosanoate (2), 7-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (3), and 6-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (4), was investigated. The study was extended to the assessment of the mode of induction of apoptosis by DCB and compound 1. The resazurin reduction assay was used for cytotoxicity studies. Assessments of cell cycle distribution, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed by flow cytometry. Constituents of DCB were isolated by column chromatography. Triterpenoid 1 and flavone 4 had cytotoxic effects towards the 9 tested cancer cell lines with IC50 values below 50 µM. The recorded IC50 values varied from 7.65 µM (towards multidrug-resistant CEM-ADR5000 leukemia cells) to 44.17 µM (against HepG2 hepatocarcinoma cells) for 1, 18.90 µM (CCRF-CEM leukemia cells) to 88.86 µM (against HCT116p53+/+ colon adenocarcinoma cells) for 4, and 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin. DCB induced apoptosis in CCRF-CEM cells mostly mediated by MMP alteration and enhanced ROS production; compound 1 induced apoptosis through caspases activation and MMP alteration and increased ROS production. Dichrostachys cinerea is an interesting cytotoxic plant and deserves more studies leading to new antineoplastic agents to fight cancer and mostly leukemia.

Fractions and isolated compounds from Oxyanthus speciosus subsp. stenocarpus (Rubiaceae) have promising antimycobacterial and intracellular activity.

BACKGROUND: Tuberculosis is a deadly disease caused by Mycobacterium species. The use of medicinal plants is an ancient global practice for the treatment and prevention of diverse ailments including tuberculosis. The aim of this study was to isolate and characterize antimycobacterial compounds by bioassay-guided fractionation of the acetone leaf extract of Oxyanthus speciosus. METHODS: A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against mycobacteria. Cytotoxicity and nitric oxide inhibitory activity of the isolated compounds was determined to evaluate in vitro safety and potential anti-inflammatory activity. Intracellular efficacy of the crude extract against Mycobacterium-infected macrophages was also determined. RESULTS: Two compounds were isolated and identified as lutein (1) and rotundic acid (2). These had good antimycobacterial activity against the four mycobacteria tested with MIC values ranging from 0.013 to 0.1 mg/mL. Rotundic acid had some cytotoxicity against C3A human liver cells. Lutein was not cytotoxic at the highest tested concentration (200 µg/mL) and inhibited nitric oxide production in RAW 264.7 macrophages by 94% at a concentration of 25 µg/mL. The acetone crude extract (120 µg/mL) of O. speciosus had intracellular antimycobacterial activity, reducing colony forming units by more than 90%, displaying bactericidal efficacy in a dose and time-dependent manner. CONCLUSION: This study provides good proof of the presence of synergism between different compounds in extracts and fractions. It is also the first report of the antimycobacterial activity of lutein and rotundic acid isolated from Oxyanthus speciosus. The promising activity of the crude extract of O. speciosus both in vitro and intracellularly in an in vitro macrophage model suggests its potential for development as an anti- tuberculosis (TB) herbal medicine.

Three New Abietane-Type Diterpenoids from Plectranthus africanus and Their Antibacterial Activities.

Three new abietane-type diterpenoids, plectranthroyleanones A - C (1:  - 3: ), together with five known compounds (4:  - 8: ) were isolated from the methanol extract of the whole plant of Plectranthus africanus using column chromatography techniques. The structures of the new compounds were elucidated using a combination of 1D and 2D NMR and HRESIMS analyses. Compound 1: exhibited weak activities with minimal inhibitory concentration values of 75 µg/mL against gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, and 150 µg/mL against two gram-negative bacteria, Pseudomonas aeruginosa and Klebsiella pneumoniae, respectively, while 2: and 3: had moderate antibacterial activity against K. pneumoniae with a minimal inhibitory concentration value of 37.5 µg/mL.

Tricalycoside, a New Cerebroside from Tricalysia coriacea (Rubiaceae).

A new cerebroside, named as tricalycoside (1), was isolated from the CH2 Cl2 /MeOH (1:1) extract of twigs and leaves of Tricalysia coriacea using repeated silica gel open column chromatography followed by preparative TLC and Sephadex LH-20, together with six known compounds (2 - 7). The structure of the new compound was determined by analysis of 1D- and 2D-NMR, MS data, chemical conversion, and by comparison of these data with those from the literature. Tricalycoside (1) possessed a weak antibacterial activity against Klebsiella pneumoniae (MIC = 75 µg/mL).

A New Benzophenanthridine Alkaloid from Caloncoba glauca.

Three alkaloids including a new one, N-formyldihydrochelerythrine (1), together with four other known compounds were isolated from the stem bark of Caloncoba glauca. The structure of the new compound was elucidated from spectroscopic and mass spectrometric evidence. This is the first report of alkaloids from the genus Caloncoba. Sesamin (4) [MIC = 256 µg/mL (Candida albicans) and dihydrochelerythrine (2) [MIC = 32 (C. albicans and C. parapsilosis), and 128 µg/mL (C. krusei)] had moderate to weak antifungal activity.

Anti-inflammatory and acetylcholinesterase activity of extract, fractions and five compounds isolated from the leaves and twigs of Artemisia annua growing in Cameroon.

BACKGROUND: Natural products, including those derived from higher plants have, over the years, contributed greatly to the development of modern therapeutic drugs. Due to the medicinal importance in traditional practice and the diversified biology and chemistry of the constituents from Artemisia spp., the genus has been receiving growing attention. The aim of this study was to investigate the ability of the ethanol extract, four fractions (F1-F4) and five compounds namely artemisinin (1), scopoletin (2), chrysosplenetin (3), eupatin (4) and 3-O-ß-d-glucopyranoside of sitosterol (5) isolated from A. annua to modulate the activity of anticholinesterase (AchE) and the production of nitric oxide (NO) in LPS-activated RAW 264.7 macrophages. RESULTS: At the lowest concentration tested (6.25 µg/mL), the crude extract and fraction F2 had the highest NO inhibitory activity (72.39 and 71.00 % inhibition respectively) without significant toxicity on the viability of macrophage cells (93.86 and 79.87 % of cell viability respectively). The crude extract inhibited AchE activity by 71.83 % (at 1 mg/mL) with an IC50 value of 87.43 µg/mL while F2 and F4 were the most active fractions (IC50 values of 36.75 and 28.82 µg/mL). Artemisinin (1) and chrysosplenetin (3) had the highest AChE activity with 71.67 and 80.00 % inhibition (at 0.1 mg/mL) and IC50 values of 29.34 and 27.14 µg/mL, respectively. CONCLUSION: Our results validate the traditional use of A. annua and could help to support the usefulness of this plant in the treatment of inflammatory and neurological disorders especially where nitric oxide and a cholinesterase are involved.

A New Alkenylmethylresorcinol from the Fruits of Ardisia kivuensis.

The phytochemical study of the MeOH extract from the fruits of Ardisia kivuensis was carried out using repeated silica gel column chromatography followed by Sephadex LH-20 to afford a new alkenylmethylresorcinol, ardisinol III (1) along with three known compounds, oleanolic acid, ß-sitosterol and pentacosanoic acid. The structure of 1 was elucidated using spectroscopic analysis (NMR and MS), and comparison with published data. Compound 1 had weak antioxidant activity (IC50 109.8 µg/mL) while other compounds were not active as compared to L-ascorbic acid (IC50 3.9 µg/mL).

A Novel Ellagic Acid Derivative from Desbordesia glaucescens.

One novel ellagic acid derivative, desglauside (1), was isolated from the leaves of Desbordesia glaucescens together with three known compounds [3',4'-di-O-methylellagic acid (2), oleanolic acid (3) and ß-sitosterol-3-O-ß-D-glucopyranoside (4)]. Their structures were elucidated on the basis of NMR spectroscopic and MS analysis, and by comparison with related published data. The crude extract, fractions and isolated compounds showed no activity against four yeast strains [Candida albicans (ATCC 9002), C. parapsilopsis (ATCC22019), C. tropicalis (ATCC750), Cryptococcus neoformans (IP95026) and one isolate of Candida guilliermondii].

Antibacterial activities of the methanol extracts, fractions and compounds from Fagara tessmannii.

ETHNOPHARMACOLOGICAL RELEVANCE: Fagara tessmannii is a shrub of the African rainforests used to treat bacterial infections, cancers, swellings and inflammation. In the present study, the methanol extract from the leaves (FTL), bark (FTB), and roots (FTR) of this plant as well as fractions (FTR1-5) and compounds isolated from FTR namely ß-sitosterol-3-O-ß-d-glucopyranoside (1), nitidine chloride (2) and buesgenine (3), were tested for their antimicrobial activities against a panel of Gram-negative bacteria including multidrug resistant (MDR) phenotypes. MATERIALS AND METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the above samples; Column chromatography was used for the fractionation and purification of the roots extract whilst the chemical structures of compounds were determined using spectroscopic techniques. RESULTS: Results of the MIC determinations indicated that the crude extracts from the roots as well as fraction FTRa4 were active on all the 26 tested bacterial strains. MIC values below 100µg/mL were obtained with roots, leaves and bark extract respectively against 30.8%, 15.4% and 11.5% tested bacteria. The lowest MIC value below of 8µg/mL was obtained with extract from the roots against Escherichia coli MC100 strain. The lowest MIC value of 4µg/mL was also obtained with compound 3 against E. coli AG102 and Klebsiella pneumoniae ATCC11296 CONCLUSIONS: The present study demonstrates that F. tessmannii is a potential source of antimicrobial drugs to fight against MDR bacteria. Benzophenanthrine alkaloids 2 and 3 are the main antibacterial consituents of the roots of the plant.

The potential role of GLUT4 transporters and insulin receptors in the hypoglycaemic activity of Ficus lutea acetone leaf extract.

BACKGROUND: Some Ficus species have been used in traditional African medicine in the treatment of diabetes. The antidiabetic potential of certain species has been confirmed in vivo but the mechanism of activity remains uncertain. The aim was to investigate the hypoglycaemic potential of ten Ficus species focussing on glucose uptake, insulin secretion and the possible mechanism of hypoglycaemic activity. METHODS: The dried and ground leaves of ten Ficus species were extracted with acetone. The dried acetone extract was reconstituted with DMSO to a concentration of 100 mg/ml which was then serially diluted and used to assay for glucose uptake in muscle, fat and liver cells, and insulin secretion in pancreatic cells. RESULTS: Only the F. lutea extract was able to modulate glucose metabolism. In comparison to insulin in the primary muscle cells, the glucose uptake ability of the extract was 33% as effective. In the hepatoma cell line, the extract was as effective as metformin in decreasing extracellular glucose concentration by approximately 20%. In the pancreatic insulin secretory assay, the extract was 4 times greater in its secretory activity than commercial glibenclamide. With F. lutea extract significantly increasing glucose uptake in the primary muscle cells, primary fat cells, C2C12 muscle and H-4-II-E liver cells, the extract may act by increasing the activity of cell surface glucose transporters. When the 3T3-L1 pre-adipocytes were compared to the primary muscle, primary fat and C2C12 cells, the differences in the former's ability to transport glucose into the cell may be due to the absence of the GLUT4 transporter, which on activation via the insulin receptor decreases extracellular glucose concentrations. Because the pre-adipocytes failed to show any active increase in glucose uptake, the present effect has to be linked to the absence of the GLUT4 transporter. CONCLUSION: Only F. lutea possessed substantial in vitro activity related to glucose metabolism. Based on the effect produced in the various cell types, F. lutea also appears to be a partial agonist/antagonist of the insulin cell membrane receptor. While the clinical effectiveness of F. lutea is not known, this plant species does possess the ability to modify glucose metabolism.

Anticandidal activity of cell extracts from 13 probiotic Lactobacillus strains and characterisation of lactic acid and a novel fatty acid derivative from one strain.

This study investigated the anti-Candida activity of methanol extracts from freeze-dried probiotic cells and the isolation of some constituents in the extracts. The MIC values of the probiotic methanol cell extracts against Candida albicans ranged between 1.25 and 5mg/ml after 48 h of incubation. However, Lactococcus latics subsp. lactis strain X and Lactobacillus casei strain B extracts had an MIC of 10mg/ml after 48 h of incubation. The extracts had fungistatic rather than fungicidal activity. These extracts had a much higher antifungal activity than antifungal compounds isolated from the growth medium by many other authors. This indicates that probiotics may also release antifungal compounds in their cells that could contribute to a therapeutic effect. Lactic acid (1) and 6-O-(α-D-glucopyranosyl)-1,6-di-O-pentadecanoyl-α-D-glucopyranose a novel fatty acid derivative (2) were isolated from methanol probiotic extracts and the structure of these compounds were elucidated using NMR (1 and 2D) and mass spectrometry (MS).

Antimicrobial activity and cytotoxicity of the ethanol extract, fractions and eight compounds isolated from Eriosema robustum (Fabaceae).

BACKGROUND: The aim of this study was to evaluate the antimicrobial activity and the cytotoxicity of the ethanol crude extract, fractions and isolated compounds from the twigs of Eriosema robustum, a plant used for the treatment of coughs and skin diseases. METHODS: Column chromatographic and spectroscopic techniques were used to isolate and identify eight compounds, robusflavones A (1) and B (2), orostachyscerebroside A (3), stigmasterol (4), 1-O-heptatriacontanoyl glycerol (5), eicosanoic acid (6), 3-O-ß-D-glucopyranoside of sitosterol (7) and 6-prenylpinocembrin (8), from E. robustum. A two-fold serial microdilution method was used to determine the minimum inhibitory concentration (MIC) against fungi and bacteria, and the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay was used to evaluate the cytotoxicity. RESULTS: Fraction B had significant antimicrobial activity against Aspergillus fumigatus and Cryptoccocus neoformans (MIC 0.08 mg/ml), whilst the crude extract and fraction A had moderate activity against A. fumigatus and Candida albicans (MIC 0.16 mg/ml). Fraction A however had excellent activity against Staphylococcus aureus (MIC 0.02 mg/ml), Enterococcus faecalis and Escherichia coli (MIC 0.04 mg/ml). The crude extract had significant activity against S. aureus, E. faecalis and E. coli. Fraction B had good activity against E. faecalis and E. coli (MIC 0.08 mg/ml). All the isolated compounds had a relatively weak antimicrobial activity. An MIC of 65 µg/ml was obtained with robusflavones A (1) and B (2) against C. albicans and A. fumigatus, orostachyscerebroside A (3) against A. fumigatus, and robusflavone B (2) against C. neoformans. Compound 8 had the best activity against bacteria (average MIC 55 µg/ml). The 3 fractions and isolated compounds had LC50 values between 13.20 to > 100 µg/ml against Vero cells yielding selectivity indices between 0.01 and 1.58. CONCLUSION: The isolated compounds generally had a much lower activity than expected based on the activity of the fractions from which they were isolated. This may be the result of synergism between different compounds in the complex extracts or fractions. The results support the traditional use of E. robustum to treat infections. The crude extract had a good activity and low preparation cost, and may be useful in topical applications to combat microbial infections.

Semi-synthesis of dihydrochalcone derivatives and their in vitro antimicrobial activities.

We describe the semi-synthesis of dihydrochalcone derivatives and their IN VITRO antimicrobial activities. These compounds were prepared by modifying two naturally occurring antimicrobial dihydrochalcones, erioschalcones A and B, reported by us earlier. The structures of the compounds were assigned on the basis of spectroscopic evidence and by comparing their physical and spectroscopic data with those reported in the literature. All the compounds were subjected to IN VITRO antimicrobial assays against a panel of pathogenic microorganisms, including gram-positive and gram-negative bacteria, and fungi. The antimicrobial efficacies of this class of compounds were established by correlating the activity profile of each compound with its structure and by comparing the activities of all the compounds with each other based on their structure. This should enable the development of other derivatives of the dihydrochalcone family that would serve as more potent antimicrobial agents against specific pathogens.

Antimicrobial prenylated dihydrochalcones from Eriosema glomerata.

Two new natural dihydrochalcones exhibiting antimicrobial properties together with six known compounds were isolated from the Cameroonian medicinal plant Eriosema glomerata. The structures of the new dihydrochalcones were elucidated as 2',4'-dihydroxy-4-methoxy-3'-( gamma, gamma-dimethylallyl)dihydrochalcone and 2',4'-dihydroxy-3'-( gamma, gamma-dimethylallyl)dihydrochalcone by detailed spectroscopic analysis. The two new dihydrochalcones, named erioschalcones A ( 1) and B ( 2), demonstrated significant inhibitory activity against the microbial strains Bacillus megaterium, Escherichia coli, Chlorella fusca and Microbotryum violaceum.