Selective Inhibition of Human Monoamine Oxidase B by Acacetin 7-Methyl Ether Isolated from Turnera diffusa (Damiana).

The investigation of the constituents that were isolated from Turnera diffusa (damiana) for their inhibitory activities against recombinant human monoamine oxidases (MAO-A and MAO-B) in vitro identified acacetin 7-methyl ether as a potent selective inhibitor of MAO-B (IC50 = 198 nM). Acacetin 7-methyl ether (also known as 5-hydroxy-4', 7-dimethoxyflavone) is a naturally occurring flavone that is present in many plants and vegetables. Acacetin 7-methyl ether was four-fold less potent as an inhibitor of MAO-B when compared to acacetin (IC50 = 50 nM). However, acacetin 7-methyl ether was >500-fold selective against MAO-B over MAO-A as compared to only two-fold selectivity shown by acacetin. Even though the IC50 for inhibition of MAO-B by acacetin 7-methyl ether was ~four-fold higher than that of the standard drug deprenyl (i.e., SelegilineTM or ZelaparTM, a selective MAO-B inhibitor), acacetin 7-methyl ether's selectivity for MAO-B over MAO-A inhibition was greater than that of deprenyl (>500- vs. 450-fold). The binding of acacetin 7-methyl ether to MAO-B was reversible and time-independent, as revealed by enzyme-inhibitor complex equilibrium dialysis assays. The investigation on the enzyme inhibition-kinetics analysis with varying concentrations of acacetin 7-methyl ether and the substrate (kynuramine) suggested a competitive mechanism of inhibition of MAO-B by acacetin 7-methyl ether with Ki value of 45 nM. The docking scores and binding-free energies of acacetin 7-methyl ether to the X-ray crystal structures of MAO-A and MAO-B confirmed the selectivity of binding of this molecule to MAO-B over MAO-A. In addition, molecular dynamics results also revealed that acacetin 7-methyl ether formed a stable and strong complex with MAO-B. The selective inhibition of MAO-B suggests further investigations on acacetin 7-methyl as a potential new drug lead for the treatment of neurodegenerative disorders, including Parkinson's disease.

Inhibition of human monoamine oxidase A and B by flavonoids isolated from two Algerian medicinal plants.

BACKGROUND: Monoamine oxidases (MAOs) are outer mitochondrial membrane flavoenzymes. They catalyze the oxidative deamination of a variety of neurotransmitters. MAO-A and MAO-B may be considered as targets for inhibitors to treat neurodegenerative diseases and depression and for managing symptoms associated with Parkinson's and Alzheimer's diseases. PURPOSE: The objective was to evaluate the inhibitory effect of Hypericum afrum and Cytisus villosus against MAO-A and B and to isolate the compounds responsible for the MAO-inhibitory activity. METHODS: The inhibitory effect of extracts and purified constituents of H. afrum and C. villosus were investigated in vitro using recombinant human MAO-A and B, and through bioassay-guided fractionation of ethyl acetate fractions of areal parts of the two plants collected in northeastern Algeria. In addition, computational protein-ligand docking and molecular dynamics simulations were carried out to explain the MAO binding at the molecular level. RESULTS: The ethyl acetate (EtOAc) fractions of H. afrum and C. villosus showed the highest MAO inhibition activity against MAO A and B with IC50 values of 3.37 µg/ml and 13.50 µg/ml as well as 5.62 and 1.87 µg/ml, respectively. Bioassay-guided fractionation of the EtOAc fractions resulted in the purification and identification of the known flavonoids quercetin, myricetin, genistein and chrysin as the principal MAO-inhibitory constituents. Their structures were established by extensive 1 and 2D NMR studies and mass spectrometry. Quercetin, myricetin and chrysin showed potent inhibitory activity towards MAO-A with IC50 values of 1.52, 9.93 and 0.25 µM, respectively, while genistein more efficiently inhibited MAO-B (IC50 value: 0.65 µM). The kinetics of the inhibition and the study of dialysis dissociation of the complex of quercetin and myricetin and the isoenzyme MAO-A showed competitive and mixed inhibition, respectively. Both compounds showed reversible binding. Molecular docking experiments and molecular dynamics simulations allowed to estimate the binding poses and to identify the most important residues involved in the selective recognition of molecules in the MAOs enzymatic clefts. CONCLUSION: Quercetin and myricetin isolated from H. afrum together with genistein and chrysin isolated from C. villosus have been identified as potent MAO-A and -B inhibitors. H. afrum and C. villosus have properties indicative of potential neuroprotective ability and may be new candidates for selective MAO-A and B inhibitors.

Isolation and Biological Evaluation of Prenylated Flavonoids from Maclura pomifera.

Phytochemical analysis of the ethanolic extract of Maclura pomifera fruits yielded four new compounds (I-IV) along with eleven known compounds (V-XV). The crude extract exhibited significant activity towards cannabinoid receptors (CB1: 103.4% displacement; CB2: 68.8% displacement) and possibly allosteric interaction with δ and µ opioid receptors (-49.7 and -53.8% displacement, resp.). Compound I was found to be possibly allosteric for κ and µ opioid receptors (-88.4 and -27.2% displacement, resp.) and showed moderate activity (60.5% displacement) towards CB1 receptor. Compound II exhibited moderate activity towards cannabinoid receptors CB1 and CB2 (47.9 and 42.3% displacement, resp.). The known compounds (V-VIII) exhibited prominent activity towards cannabinoid receptors: pomiferin (V) (IC50 of 2.110 and 1.318 µM for CB1 and CB2, resp.), auriculasin (VI) (IC50 of 8.923 µM for CB1), warangalone (VII) (IC50 of 1.670 and 4.438 µM for CB1 and CB2, resp.), and osajin (VIII) (IC50 of 3.859 and 7.646 µM for CB1 and CB2, resp.). The isolated compounds were also tested for inhibition of human monoamine oxidase-A and monoamine oxidase-B enzymes activities, where all the tested compounds showed fewer inhibitory effects on MAO-A compared to MAO-B activities: auriculasin (VI) (IC50 of 1.91 and 45.98 µM for MAO-B and MAO-A, resp.).

Monoamine oxidases inhibitors from Colvillea racemosa: Isolation, biological evaluation, and computational study.

Bioassay-guided fractionation and chemical investigation of Colvillea racemosa stems led to identification of two new α, ß-dihydroxydihydrochalcones, colveol A (1) and colveol B (2) along with fifteen known compounds. The structures were elucidated via interpretation of spectroscopic data. The absolute configurations of the dihydrochalcones 1 and 2 were assigned by a combination of chemical modification and electronic circular dichroism data. The isolated compounds were evaluated for their inhibition activity toward recombinant human monoamine oxidases (rhMAO-A and -B). Compound 1 demonstrated preferential inhibition against hMAO-A isoenzyme (IC50 0.62µM, SIA/B 0.02) while S-naringenin (13) and isoliquiritigein (15) demonstrated preferential hMAO-B inhibition (IC50 0.27 and 0.51µM, SIA/B 31.77 and 44.69, respectively). Fisetin (11) showed inhibition against hMAO-A with IC50 value of 4.62µM and no inhibitory activity toward hMAO-B up to 100µM. Molecular docking studies for the most active compounds were conducted to demonstrate the putative binding modes. It suggested that 1 interacts with Gln215, Ala111, Phe352, and Phe208 amino acid residues which have a role in the orientation and stabilization of the inhibitor binding to hMAO-A, while S-naringenin (13) occupies both entrance and substrate cavities and interacts with Tyr326, a critical residue in inhibitor recognition in hMAO-B.

Interactions of Desmethoxyyangonin, a Secondary Metabolite from Renealmia alpinia, with Human Monoamine Oxidase-A and Oxidase-B.

Renealmia alpinia (Zingiberaceae), a medicinal plant of tropical rainforests, is used to treat snakebites and other injuries and also as a febrifuge, analgesic, antiemetic, antiulcer, and anticonvulsant. The dichloromethane extract of R. alpinia leaves showed potent inhibition of human monoamine oxidases- (MAOs-) A and B. Phytochemical studies yielded six known compounds, including pinostrobin 1, 4'-methyl ether sakuranetin 2, sakuranetin 3, pinostrobin chalcone 4, yashabushidiol A 5, and desmethoxyyangonin 6. Compound 6 displayed about 30-fold higher affinity for MAO-B than MAO-A, with Ki values of 31 and 922 nM, respectively. Kinetic analysis of inhibition and equilibrium-dialysis dissociation assay of the enzyme-inhibitor complex showed reversible binding of desmethoxyyangonin 6 with MAO-A and MAO-B. The binding interactions of compound 6 in the active site of the MAO-A and MAO-B isoenzymes, investigated through molecular modeling algorithms, confirmed preferential binding of desmethoxyyangonin 6 with MAO-B compared to MAO-A. Selective reversible inhibitors of MAO-B, like desmethoxyyangonin 6, may have important therapeutic significance for the treatment of neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease.

A Combined In Vitro Assay for Evaluation of Neurotrophic Activity and Cytotoxicity.

Neurotrophic assays are phenotypic methods to identify molecules that stimulate differentiation of neuronal cells. Bioactive small molecules with neurotrophic actions hold great promise as therapeutic agents for the treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth. A combined in vitro method, which measures neurotrophic activity and cytotoxicity in a single assay, has been described. This assay, performed in 96-well microplates with PC12 and Neuroscreen-1 (NS-1; a subclone of PC12) cells, is a simple tool for identification of new neurotrophic agents. Stimulation of neurite outgrowth was measured with NIS software by analysis of digital cell images as multiple parameters, namely, mean neurite length, neurite length/cell, nodes/cell, and number of neurites/cell. The assay has been standardized and validated with dose-response analysis for nerve growth factor (NGF) and mechanism-based inhibitors of NGF-induced neurite outgrowth, namely, SU6656 (an Src family kinase inhibitor) and PD98059 (a MEK inhibitor). The assay has been successfully applied for screening natural and synthetic compound libraries for cytotoxicity and neurotrophic activity. Screening of a set of harmala alkaloids identified harmine as a potential neurotrophic molecule that significantly stimulated NGF-induced neurite outgrowth in the NS-1 cells. Important advantages of this method are its simplicity and determination of cytotoxicity and neurotrophic activity in a single assay. This assay may be suitable for primary and cultured neuronal cells.

Isolation of Acacetin from Calea urticifolia with Inhibitory Properties against Human Monoamine Oxidase-A and -B.

Calea urticifolia (Asteraceae: Asteroideae) has long been used as a traditional medicine in El Salvador to treat arthritis and fever, among other illnesses. The chloroform extract of the leaves of C. urticifolia showed potent inhibition of recombinant human monoamine oxidases (MAO-A and -B). Further bioassay-guided fractionation led to the isolation of a flavonoid, acacetin, as the most prominent MAO inhibitory constituent, with IC50 values of 121 and 49 nM for MAO-A and -B, respectively. The potency of MAO inhibition by acacetin was >5-fold higher for MAO-A (0.121 µM vs 0.640 µM) and >22-fold higher for MAO-B (0.049 µM vs 1.12 µM) as compared to apigenin, the closest flavone structural analogue. Interaction and binding characteristics of acacetin with MAO-A and -B were determined by enzyme-kinetic assays, enzyme-inhibitor complex binding, equilibrium-dialysis dissociation analyses, and computation analysis. Follow-up studies showed reversible binding of acacetin with human MAO-A and -B, resulting in competitive inhibition. Acacetin showed more preference toward MAO-B than to MAO-A, suggesting its potential for eliciting selective pharmacological effects that might be useful in the treatment of neurological and psychiatric disorders. In addition, the binding modes of acacetin at the enzymatic site of MAO-A and -B were predicted through molecular modeling algorithms, illustrating the high importance of ligand interaction with negative and positive free energy regions of the enzyme active site.

Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice.

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10-100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3-30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.

Composition, standardization and chemical profiling of Banisteriopsis caapi, a plant for the treatment of neurodegenerative disorders relevant to Parkinson's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Banisteriopsis caapi, a woody vine from the Amazonian basin, is popularly known as an ingredient of a sacred drink ayahuasca, widely used throughout the Amazon as a medicinal tea for healing and spiritual exploration. The usefulness of Banisteriopsis caapi has been established for alleviating symptoms of neurological disorders including Parkinson's disease. AIM OF THE STUDY: Primary objective of this study was to develop the process for preparing standardized extracts of Banisteriopsis caapi to achieve high potency for inhibition of human monoamine oxidases (MAO) and antioxidant properties. The aqueous extracts prepared from different parts of the plant collected from different geographical locations and seasons were analyzed by HPLC for principal bioactive markers. The extracts were simultaneously tested in vitro for inhibition of human MAOs and antioxidant activity for analysis of correlation between phytochemical composition of the extracts and bioactivities. MATERIALS AND METHODS: Reversed-phase HPLC with photodiode array detection was employed to profile the alkaloidal and non-alkaloidal components of the aqueous extract of Banisteriopsis caapi. The Banisteriopsis caapi extracts and standardized compositions were tested in vitro for inhibition of recombinant preparations of human MAO-A and MAO-B. In vitro cell-based assays were employed for evaluation of antioxidant property and mammalian cell cytotoxicity of these preparations. RESULTS: Among the different aerial parts, leaves, stems/large branches and stem bark of Banisteriopsis caapi, HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7-9) were present in high concentrations in dried bark of large branch. A library of HPLC chromatograms has also been generated as a tool for fingerprinting and authentication of the studied Banisteriopsis caapi species. The correlation between potency of MAO inhibition and antioxidant activity with the content of the main active constituents of the aqueous Banisteriopsis caapi extracts and standardized compositions was established. Phytochemical analysis of regular/commercial Banisteriopsis caapi dried stems, obtained from different sources, showed a similar qualitative HPLC profile, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency compared to Banisteriopsis caapi Da Vine. CONCLUSION: The ethnopharmacological use of bark of matured stem/large branch of Banisteriopsis caapi as well as whole matured stem is supported by the results obtained in this investigation. Among various constituents of Banisteriopsis caapi, harmine (7), harmaline (6) and tetrahydroharmine (5) are responsible for MAO-A inhibition, while two major proanthocyanidines, epicatechin (8) and procyanidine B2 (9) produce antioxidant effects. The compounds 1-9 can serve as reliable markers for identification and standardization of Banisteriopsis caapi aerial parts, collected in different seasons and/or from different geographical regions.

Metabolic clustering of a core collection of Indian ginseng Withania somnifera Dunal through DNA, isoenzyme, polypeptide and withanolide profile diversity.

Withania somnifera is one of the most important medicinal plants of Ayurveda and finds extensive uses in Indian traditional herbal preparations. In this investigation, selected accessions of the plant were examined for diversity through RAPDs, isoenzymes, polypeptide polymorphism and withanolide profiles. The accessions clustered together with respect to their characteristic profile of major withanolides and represented withaferin A, withanone, withanolide D or withanolide A rich groups. This level of phytochemical diversity as discrete chemotypes is widest and is being first ever documented to occur in Indian population of the plant.