Neurotoxic Zanthoxylum chalybeum root constituents invoke mosquito larval growth retardation through ecdysteroidogenic CYP450s transcriptional perturbations.

Intracellular effects exerted by phytochemicals eliciting insect growth-retarding responses during vector control intervention remain largely underexplored. We studied the effects of Zanthoxylum chalybeum Engl. (Rutaceae) (ZCE) root derivatives against malaria (Anopheles gambiae) and arbovirus vector (Aedes aegypti) larvae to decipher possible molecular targets. We report dose-dependent biphasic effects on larval response, with transient exposure to ZCE and its bioactive fraction (ZCFr.5) inhibiting acetylcholinesterase (AChE) activity, inducing larval lethality and growth retardation at sublethal doses. Half-maximal lethal concentrations (LC50) for ZCE against An. gambiae and Ae. aegypti larvae after 24-h exposure were 9.00 ppm and 12.26 ppm, respectively. The active fraction ZCFr.5 exerted LC50 of 1.58 ppm and 3.21 ppm for An. gambiae and Ae. aegypti larvae, respectively. Inhibition of AChE was potentially linked to larval toxicity afforded by 2-tridecanone, palmitic acid (hexadecanoic acid), linoleic acid ((Z,Z)-9,12-octadecadienoic acid), sesamin, ß-caryophyllene among other compounds identified in the bioactive fraction. In addition, the phenotypic larval retardation induced by ZCE root constituents was exerted through transcriptional modulation of ecdysteroidogenic CYP450 genes. Collectively, these findings provide an explorative avenue for developing potential mosquito control agents from Z. chalybeum root constituents.

Inhibition Kinetics and Theoretical Studies on Zanthoxylum chalybeum Engl. Dual Inhibitors of α-Glucosidase and α-Amylase.

Compounds from Zanthoxylum chalybeum Engl. were previously reported for inhibitory activities of amylase and glucosidase enzymatic action on starch as a preliminary study toward the establishment of a management strategy against postprandial hyperglycemia, however, the inhibitory kinetics and molecular interaction of these compounds were never established. A study was thus designed to establish the inhibitory kinetics and in silico molecular interaction of α-glucosidase and α-amylase with Z. chalybeum metabolites based on Lineweaver-Burk/Dixon plot analyses and using Molecular Operating Environment (MOE) software, respectively. Skimmianine (5), Norchelerythrine (6), 6-Acetonyldihydrochelerythrine (7), and 6-Hydroxy-N-methyldecarine (8) alkaloids showed mixed inhibition against both α-glucosidase and α-amylase with comparable Ki to the reference acarbose (p > 0.05) on amylase but significantly higher activity than acarbose on α-glucosidase. One phenolic 2,3-Epoxy-6,7-methylenedioxyconiferol (10) showed a competitive mode of inhibition both on amylase and glucosidase which were comparable (p > 0.05) to the activity of acarbose. The other compounds analyzed and displayed varied modes of inhibition between noncompetitive and uncompetitive with moderate inhibition constants included chaylbemide A (1), chalybeate B (2) and chalybemide C (3), fagaramide (4), ailanthoidol (9), and sesame (11). The important residues of the proteins α-glucosidase and α-amylase were found to have exceptional binding affinities and significant interactions through molecular docking studies. The binding affinities were observed in the range of -9.4 to -13.8 and -8.0 to -12.6 relative to the acarbose affinities at -17.6 and -20.5 kcal/mol on α-amylase and α-glucosidase residue, respectively. H-bonding, π-H, and ionic interactions were noted on variable amino acid residues on both enzymes. The study thus provides the basic information validating the application of extracts of Z. chalybeum in the management of postprandial hyperglycemia. Additionally, the molecular binding mechanism discovered in this study could be useful for optimizing and designing new molecular analogs as pharmacological agents against diabetes.

Cytotoxicity of fagaramide derivative and canthin-6-one from Zanthoxylum (Rutaceae) species against multidrug resistant leukemia cells.

In our continuous search for cytotoxic compounds from the genus Zanthoxylum, chromatographic separation of the MeOH/CH2Cl2 (1:1) extract of Z. chalybeum yielded one new alkamide; 4-(isoprenyloxy)-3-methoxy-3,4-deoxymethylenedioxyfagaramide (1) and a known one; fagaramide (2). Similarly, from the MeOH/CH2Cl2 (1:1) extract of the stem bark of Z. parachanthum four known compounds; canthin-6-one (3), dihydrochelerythrine (4), lupeol (5) and sesamin (6) were isolated. Characterization of the structures of these compounds was achieved using spectroscopic techniques (NMR and MS). Using resazurin reduction assay 1, 3 and 6 displayed moderate cytotoxicity with IC50 values below 50 µM against the drug sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cell lines. It is interesting to note that 3 was more active than the standard drug, doxorubicin against CEM/ADR5000 leukemia cells. Compounds 3 and 6 showed good selectivity on leukemia cells than normal cells. In future studies 3 should be tested against a panel of drug resistant human cells.

Antiinflammatory Medicinal Plants from the Ugandan Greater Mpigi Region Act as Potent Inhibitors in the COX-2/PGH2 Pathway.

Our study investigates 16 medicinal plants via assessment of inhibition of proinflammatory enzymes such as cyclooxygenases (COX). The plants are used by traditional healers in the Greater Mpigi region in Uganda to treat inflammation and related disorders. We present results of diverse in vitro experiments performed with 76 different plant extracts, namely, (1) selective COX-2 and COX-1 inhibitor screening; (2) 15-LOX inhibition screening; (3) antibacterial resazurin assay against multidrug-resistant Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, and Escherichia coli K12; (4) DPPH assay for antioxidant activity; and (5) determination of the total phenolic content (TPC). Results showed a high correlation between traditional use and pharmacological activity, e.g., extracts of 15 out of the 16 plant species displayed significant selective COX-2 inhibition activity in the PGH2 pathway. The most active COX-2 inhibitors (IC50 < 20 µg/mL) were nine extracts from Leucas calostachys, Solanum aculeastrum, Sesamum calycinum subsp. angustifolium, Plectranthus hadiensis, Morella kandtiana, Zanthoxylum chalybeum, and Warburgia ugandensis. There was no counteractivity between COX-2 and 15-LOX inhibition in these nine extracts. The ethyl acetate extract of Leucas calostachys showed the lowest IC50 value with 0.66 µg/mL (COX-2), as well as the most promising selectivity ratio with 0.1 (COX-2/COX-1). The TPCs and the EC50 values for DPPH radical scavenging activity showed no correlation with COX-2 inhibitory activity. This led to the assumption that the mechanisms of action are most likely not based on scavenging of reactive oxygen species and antioxidant activities. The diethyl ether extract of Harungana madagascariensis stem bark displayed the highest growth inhibition activity against S. aureus (MIC value: 13 µg/mL), L. innocua (MIC value: 40 µg/mL), and L. monocytogenes (MIC value: 150 µg/mL). This study provides further evidence for the therapeutic use of the previously identified plants used medicinally in the Greater Mpigi region.

α-Amylase and α-glucosidase inhibitors from Zanthoxylum chalybeum Engl. root bark.

A systematic analysis of the root bark of Zanthoxylum chalybeum was conducted to establish the antidiabetic potential of isolated compounds based on its ethnomedicinal use to manage diabetes. Chromatographic separation of alkaloid extracts led to isolation of three undescribed amides, chaylbemide A (1), chalybemide B (2) and chalybemide C (3) alongside the known fagaramide (4); four known benzophenanthridine alkaloids skimmianine (5), norchelerythrine (6), 6-acetonyldihydrochelerythrine (7) and 6-hydroxy-N-methyl decarine (8). The alkaloid free extracts yielded three known lignans, ailanthoidol (9), 2,3-epoxy-6,7-methylenedioxy coniferyl alcohol (10), sesamine (11), together with five known triterpenoids, lupeol (12), lupanone (13), 3α,20-dihydroxy-28-lupanoic acid (14), 20-hydroxy-3-oxo-28-lupanoic acid (15) and 3α,20,28-trihydroxylupane (16). The structures of the compounds were established based on 1D and 2D NMR spectroscopic and mass spectrometric experiments. Compounds 1-8 displayed inhibitory activities against both α-amylase and α-glycosidase in the range of IC50 = 43.22-49.36 µM which showed no significant (P > 0.05) difference to the positive control acarbose (IC50 = 42.67; 44.88 µM). The results confirmed anti-hyperglycemic potential of alkaloids from Z. chalybeum which lends credence to its use towards management of diabetes susceptibilities.

Antiplasmodial activity and phytochemical analysis of extracts from selected Ugandan medicinal plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Resistance of the parasites to known antimalarial drugs has provided the necessity to find new drugs from natural products against malaria. The aim of the study was to evaluate the in vitro antiplasmodial activity of some plants used by Traditional Medical Practitioners (TMPs) of Prometra and Rukararwe in malaria treatment in Uganda to provide scientific proof of the efficacies claimed by these Herbalists. MATERIALS AND METHODS: The air dried samples of Clerodendrum rotundifolium (leaves), Microglossa pyrifolia (leaves), Momordica foetida (leaves) and Zanthoxylum chalybeum (stem bark) used for malaria treatment by TMPs were successively extracted with ethyl acetate, methanol and water to yield twelve extracts. The extracts were tested against the chloroquine-sensitive (NF54) and chloroquine-resistant (FCR3) Plasmodium falciparum strains in vitro using the micro Mark III test which is based on assessing the inhibition of schizont maturation. A compound A was extracted and purified from the stem bark of Z. chalybeum and its structure was identified and confirmed by spectroscopic methods. RESULTS: Most of the extracts tested (92%) showed an antiplasmodial activity with IC50<50µg/mL. In spite of successive extractions with different solvents, potent anti-plasmodial activity (IC50<5µg/mL) was observed in the ethyl acetate, methanol and aqueous extracts of M. pyrifolia and C. rotundifolium. Preferential enrichments of activity into water (IC50<15µg/mL) and Ethyl acetate (IC50<5µg/mL) were seen in the case of M. foetida and Z. chalybeum respectively. The most active extracts were from C. rotundifolium and M. pyrifolia with IC50 values less than 2µg/mL. Phytochemical analysis of the extracts revealed the presence of saponins, tannins, flavonoids, alkaloids and cardiac glycocides. Fagaramide isolated from Z. chalybeum had a higher activity (IC50 2.85µg/mL) against the chloroquine-resistant strain than against the chloroquine-senstive (IC50 16.6µg/mL) strain used in the study. CONCLUSION: The plant extracts analysed in this study presented an average antiplasmodial activity (58%). This study revealed for the first time the antiplasmodial activity of the plant C. rotundofolium. It's the first time the compound fagaramide (N-isobutyl-3-(3,4-methylene dioxyphenyl)-2E-propenamide) has been isolated from Z. chalybeum as one of the compounds that contribute to the activity of this plant against P. falciparum.