Antibacterial, Antibiofilm, and Efflux Pump Inhibitory Properties of the Crude Extract and Fractions from Acacia macrostachya Stem Bark.

Microbial infections remain a public health problem due to the upsurge of bacterial resistance. In this study, the antibacterial, antibiofilm, and efflux pump inhibitory activities of the stem bark of Acacia macrostachya, an indigenous African medicinal plant, were investigated. In traditional medicine, the plant is used in the treatment of microbial infections and inflammatory conditions. A crude methanol extract obtained by Soxhlet extraction was partitioned by column chromatography to obtain the petroleum ether, ethyl acetate, and methanol fractions. Antibacterial, efflux pump inhibition and antibiofilm formation activities were assessed by the high-throughput spot culture growth inhibition (HT-SPOTi), ethidium bromide accumulation, and the crystal violet retention assay, respectively. The minimum inhibitory concentrations (MICs) of the crude extract and major fractions ranged from 250 to ≥500 µg/mL. At a concentration of 3.9-250 µg/mL, all extracts demonstrated >80% inhibition of biofilm formation in S. aureus. In P. aeruginosa, the EtOAc fraction showed the highest antibiofilm activity (59-69%) while the pet-ether fraction was most active against E. coli biofilms (45-67%). Among the test samples, the crude extract, methanol, and ethyl acetate fractions showed remarkable efflux pump inhibition in S. aureus, E. coli, and P. aeruginosa. At ½ MIC, the methanol fraction demonstrated significant accumulation of EtBr in E. coli having superior efflux inhibition over the standard EPIs: chlorpromazine and verapamil. Tannins, flavonoids, triterpenoids, phytosterols, coumarins, and saponins were identified in preliminary phytochemical studies. Stigmasterol was identified in the EtOAc fraction. This study justifies the use of A. macrostachya in the treatment of infections in traditional medicine and highlights its potential as a source of bioactive compounds that could possibly interact with some resistance mechanisms in bacteria to combat antimicrobial resistance.

Bacterial efflux inhibitors are widely distributed in land plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Secondary metabolites play a critical role in plant defense against disease and are of great importance to ethnomedicine. Bacterial efflux pumps are active transport proteins that bacterial cells use to protect themselves against multiple toxic compounds, including many antimicrobials. Efflux pump inhibitors from plants can block these efflux pumps, increasing the potency of antimicrobial compounds. This study demonstrates that efflux pump inhibition against the Gram-positive bacterial pathogen Staphylococcus aureus is widespread in extracts prepared from individual species throughout the land plant lineage. It therefore suggests a general mechanism by which plants used by indigenous species may be effective as a topical treatment for some bacterial infections. AIM OF THE STUDY: The goal of this research was to evaluate the distribution of efflux pump inhibitors in nine plant extracts with an ethnobotanical use suggestive of an antimicrobial function for the presence of efflux pump inhibitory activity against Staphylococcus aureus. MATERIALS AND METHODS: Plants were collected, dried, extracted, and vouchers submitted to the Herbarium of the University of North Carolina Chapel Hill (NCU). The extracts were analyzed by quantitative mass spectrometry (UPLC-MS) to determine the presence and concentration of flavonoids with known efflux pump inhibitory activity. A mass spectrometry-based assay was employed to measure efflux pump inhibition for all extracts against Staphylococcus aureus. The assay relies on UPLC-MS measurement of changes in ethidium concentration in the spent culture broth when extracts are incubated with bacteria. RESULTS: Eight of these nine plant extracts inhibited toxic compound efflux at concentrations below the MIC (minimum inhibitory concentration) value for the same extract. The most active extracts were those prepared from Osmunda claytoniana L. and Pinus strobes L., which both demonstrated IC50 values for efflux inhibition of 19 ppm. CONCLUSIONS: Our findings indicate that efflux pump inhibitors active against Staphylococcus aureus are common in land plants. By extension, this activity is likely to be important in many plant-derived antimicrobial extracts, including those used in traditional medicine, and evaluation of efflux pump inhibition may often be valuable when studying natural product efficacy.

Antimicrobial Peptides From Lycosidae (Sundevall, 1833) Spiders.

Antimicrobial peptides (AMPs) have been found in all organism taxa and may play an essential role as a host defense system. AMPs are organized in various conformations, such as linear peptides, disulfide bond-linked peptides, backbone-linked peptides and circular peptides. AMPs apparently act primarily on the plasma membrane, although an increasing number of works have shown that they may also target various intracellular sites. Spider venoms are rich sources of biomolecules that show several activities, including modulation or blockage of ion channels, anti-insect, anti-cancer, antihypertensive and antimicrobial activities, among others. In spider venoms from the Lycosidae family there are many linear AMPs with a wide range of activities against several microorganisms. Due to these singular activities, some Lycosidae AMPs have been modified to improve or decrease desirable or undesirable effects, respectively. Such modifications, especially with the aim of increasing their antibiotic activity, have led to the filing of many patent applications. This review explores the abundance of Lycosidae venom AMPs and some of their derivatives, and their use as new drug models.

Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

The potential of Sutherlandia frutescens for herb-drug interaction.

In Africa, Sutherlandia frutescens is a popular medicinal herb widely consumed by people living with human immunodeficiency virus/AIDS. Concomitant use with antiretroviral drugs has generated concerns of herb-drug interaction (HDI). This study investigated the inhibitory effects of the crude extracts of S. frutescens on the major cytochrome P450 isozymes with the use of pooled human liver microsomes. Its effect on the metabolic clearance of midazolam using cryopreserved hepatocytes was also monitored. The potential of S. frutescens to inhibit human ATP-binding cassette transporters (P-gp and BCRP) and the human organic anion transporting polypeptide (OATP1B1 and OATP1B3) activity was assessed using cell lines overexpressing the transporter proteins. S. frutescens showed inhibitory potency for CYP1A2 (IC(50) = 41.0 µg/ml), CYP2A6 (IC(50) = 160 µg/ml), CYP2B6 (IC(50) = 20.0 µg/ml), CYP2C8 (IC(50) = 22.4 µg/ml), CYP2C9 (IC(50) = 23.0 µg/ml), CYP2C19 (IC(50) = 35.9 µg/ml), and CYP3A4/5 (IC(50) = 17.5 µg/ml [with midazolam1'-hydroxylation]; IC(50) = 28.3 µg/ml [with testosterone 6ß-hydroxylation]). Time-dependent (irreversible) inhibition by S. frutescens was observed for CYP3A4/5 (K(I) = 296 µg/ml, k(inact) = 0.063 min(-1)) under the conditions of this study. S. frutescens also delays the production of midazolam metabolites in the hepatocytes, decreasing its clearance by 40%. Furthermore, S. frutescens inhibited P-gp (IC(50) = 324.8 µg/ml), OATP1B1 (IC(50) = 10.4 µg/ml), and OATP1B3 (IC(50) = 6.6 µg/ml). The result indicates the potential for HDI between S. frutescens and the substrates of the affected enzymes, if sufficient in vivo concentration of the extract is attained.

The effects of green tea polyphenols on drug metabolism.

INTRODUCTION: Tea, made from the dried leaves of the plant Camellia sinensis Theaceae, is a very popular beverage consumed worldwide. Recently, green tea extract-based dietary supplements have also been widely consumed for the acclaimed beneficial health effects, such as weight reduction. Although tea consumption is considered to be innocuous, the potential interactions between tea polyphenols and drugs have been demonstrated in studies in vitro and in vivo. AREAS COVERED: This article reviews the current literature on the chemistry and biotransformation of tea constituents, mainly catechins from green tea. The article also provides a review of their effects on the absorption, efflux, metabolism and elimination of different drugs. EXPERT OPINION: Tea catechins may bind to certain drugs to affect their absorption and bioactivities. Tea catechins may inhibit the activities of drug-metabolizing enzymes and drug transporters or affect the expression of these proteins, either upregulation or downregulation. Although these effects have been demonstrated in studies in vitro and in animal models, such effects have only been observed in limited cases in humans at common doses of human tea consumption. The ingestion of tea catechins from dietary supplements, which could be in large bullet doses, may produce more profound effects on drug metabolism, and such effects with drugs need to be further investigated.

Relaxant effect induced by wogonin from Scutellaria baicalensis on rat isolated uterine smooth muscle.

CONTEXT: Wogonin is a flavone derivative isolated from Scutellaria baicalensis Georgi (Labiatae) root, which is a traditional Chinese drug used as an anti-inflammatory and for management of dysmenorrhea. OBJECTIVE: The effect of wogonin on the uterus has not yet been examined. We investigated the relaxant effects of wogonin on contractile activity of isolated uterine strips of rats. MATERIALS AND METHODS: The effect of wogonin on spontaneous uterine contraction, and uterine contraction induced by agonists, K⁺-depolarization and oxytocin in Ca²âº-free solution was observed. To clarify the type of potassium channel, we tested the effects of 4-aminopyridine, tetraethylammonium and glibenclamide. RESULTS: Wogonin reduced the contractile amplitude of uterine strip smooth muscle of rats in a dose-dependent manner. The concentration of wogonin for reducing the contraction amplitude by 50% (IC50) on spontaneous contractions was 60.5 µM. Wogonin also inhibited the contraction induced by three agonists (oxytocin, prostaglandin F(2α) and acetylcholine). For the uterine strips pretreated with oxytocin in Ca²âº-free solution or K⁺-depolarization, wogonin showed relaxant effect on the induced uterine contractions. In addition, whereas the inhibitive effect of wogonin on the contraction of uterine smooth muscle in rats could be partly blocked by 4-aminopyridine and tetraethylammonium, it was not influenced by glibenclamide. DISCUSSION AND CONCLUSION: Wogonin significantly inhibited the contraction of rat uterine smooth muscle probably through the inhibition of the inflow of extracellular calcium into cells via cell membrane, and intracellular release of calcium ions. In addition, the relaxant effect induced by wogonin might be due in part to the opening of voltage-dependent and large conductance Ca²âº-activated K⁺ channels.

Isolation and structure elucidation of major alkaline degradant of ezetimibe.

This work presents the isolation and characterization of the alkaline degradant of Ezetimibe. Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, was subjected to alkaline degradation. Ezetimibe was reacted with 0.1M methanolic sodium hydroxide solution for 10min at 80°C to yield alkaline degradant to an extent of 90% of initial amount of the drug taken. This degradant was detected by high performance liquid chromatography (HPLC) at relative retention time (RRT) of 1.48 with respect to Ezetimibe. HPLC method involved an isocratic elution on a Waters Symmetry C(8) 150mm×4.6mm, 5µm column using ammonium acetate buffer (pH 4.5, 50mM) - acetonitrile (50:50, v/v) as the mobile phase at a flow rate of 1.0mL/min and UV detection at 242nm. The degradant was isolated by preparative HPLC. Purity of the isolated solid was found to be more than 99%. Structure of alkaline degradant was confirmed by LC-MS, (1)H and (13)C NMR and IR spectroscopy. On the basis of spectral data, the structure of the degradant was confirmed as 5-(4-fluorophenyl)-2-[(4-fluorophenyl amino)-(4-hydroxyphenyl)methyl]-pent-4-enoic acid. The route for the formation of this degradant is also proposed. Determining the structures of degradation products arouse during stress testing can be useful for preclinical discovery efforts.

Isolation and characterization of a high affinity peptide inhibitor of ClC-2 chloride channels.

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K(D) of approximately 20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.

Isolation and characterization of Jingzhaotoxin-V, a novel neurotoxin from the venom of the spider Chilobrachys jingzhao.

Jingzhaotoxin-V (JZTX-V), a 29-residue polypeptide, is derived from the venom of the spider Chilobrachys jingzhao. Its cDNA determined by rapid amplification of 3' and 5'-cDNA ends encoded an 83-residue precursor with a pro-region of 16 residues. JZTX-V inhibits tetrodotoxin-resistant and tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion neurons with IC50 values of 27.6 and 30.2 nM, respectively. Moreover, the toxin exhibits high affinity to the resting closed states of the channels. JZTX-V also inhibits Kv4.2 potassium currents expressed in Xenpus Laevis oocytes (IC50=604.2 nM), but has no effects on outward delay-rectified potassium channels expressed in Xenopus laevis oocytes. JZTX-V alters the gating properties of sodium channels by shifting the activation curves to the depolarizing direction and the inactivation curves to the hyperpolarizing direction. Small unilamellar vesicles binding assays show that the partitioning of JZTX-V into lipid bilayer requires negatively charged phospholipids. The phospholipid membrane binding activity of JZTX-V is also verified using intrinsic tryptophan fluorescence analysis as well as acrylamide-quenching assays. Importantly, human multiple sodium channel subtypes are attractive targets for treatment of pain, highlighting the importance of JZTX-V as potential lead for drug development.