Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Flavonoid glycosides and isoquinolinone alkaloids from Corydalis bungeana

Two flavonol O-glycosides identified as the 3-O-alpha-arabinopyranosyl(1'-->6')-beta-glucopyranoside 7-O-beta-glucopyranosides of kaempferol and quercetin were isolated from the whole plant of Corydalis bungeana Turcz. together with eight known flavonol O-glycosides. Two isoquinolinone alkaloids were also obtained from the same source, including the new derivative, 6,7-methylenedioxy-2-(6-acetyl-2,3-methylenedioxybenzyl)-1(2H)-isoquinolinone. The structures were determined by spectroscopic methods (NMR and high-resolution MS).     

Comparative study of the alkaloids in tribe Datureae and their chemosystematic significance

Sixty-six tropane alkaloids from crude leaf and root alkaloid mixtures of 12 different species and their varieties and subspecies from tribe Datureae were determined by GC–MS. The alkaloids 3β-hydroxy-6β-acetoxytropane, 3-propionyloxy-6-hyroxytropane, 3β-hydroxy-6β-tigloyloxytropane, 3β-tigloyloxy-6β-acetoxytropane and 3-tigloyloxy-7-isobutyryloxytropane are new reported tropane alkaloids.     

Characterisation of alkaloids from some Australian Stephania (Menispermaceae) species

Chemical investigations of some Stephania species native to Australia and reportedly employed by Aboriginal people as therapeutic agents, are described. The alkaloids from the forest vines Stephania bancroftii F.M. Bailey and S. aculeata F.M. Bailey (Menispermaceae) have been isolated and characterised. The major alkaloids in the tuber of the former species are (-)-tetrahydropalmatine and (-)-stephanine, whereas these are minor components in the leaves, from which a C-7 hydroxylated aporphine has been identified. The major tuber alkaloids in S. aculeata are (+)-laudanidine, and the morphinoid, (-)-amurine, whose absolute stereochemistry has been established by X-ray structural analysis of the methiodide derivative. No significant levels of alkaloids were detected in S. japonica. Complete and unambiguous 1H and 13C NMR data are presented for these alkaloids.     

Alkaloids in the venom of Messor ants

Tobacco alkaloids of the anabasine type have been found or confirmed in the venom of five species of arid-dwelling Messor ants. They are frequently accompanied by alkylpyrazines. Messor mediorubra contains four alkaloids, with anabasine the major component and also minor pyrazines. Anabasine was found alone in the venom of Messor semirufus and confirmed in Messor ebeninus. Messor rugosus from Tel Aviv contained a mixture of alkaloids and pyrazines, but those from Ein Yahav contained 3-ethyl-2,5-dimethylpyrazine, some 2-phenylethylamine, and N-ethylidene 2-phenylethylamine. Messor arenarius is confirmed as having a complex but variable mixture of alkaloids and pyrazines.     

Chemotaxonomy of Portuguese Ulex: quinolizidine alkaloids as taxonomical markers

Six species of Portuguese Ulex L. in a total of nineteen populations were studied by GC-EIMS as to their content in quinolizidine alkaloids. Sparteine, beta-isosparteine, jussiaeiine A, N-methylcytisine, cytisine, 5,6-dehydrolupanine, rhombifoline, lupanine, jussiaeiine B, N-formylcytisine, N-acetylcytisine, anagyrine, jussiaeiine C, jussiaeiine D, pohakuline, baptifoline, and epibaptifoline were detected. Analysis of the chromatograms showed that the chemical profile of all species was mainly composed of N-methylcytisine, cytisine, anagyrine, and jussiaeiines A, B, C and D. Therefore a quantification study of these alkaloids in all the populations studied was done by GC. These data were then submitted to cluster analysis and principal component analysis, which allowed the definition of five chemotypes and the recognition of hybrids. N-methylcytisine, cytisine, and jussiaeiines A, C and D are recognized as markers of this genus in Portugal.     

E/Z-Thesinine-O-4'-alpha-rhamnoside, pyrrolizidine conjugates produced by grasses (Poaceae)

Based on direct infusion mass spectrometry we identified a novel alkaloid as a major component of perennial ryegrass (Lolium perenne). Initial mass spectral data suggested it to be a pyrrolizidine conjugate. As this class of alkaloids has not been described before from grasses, we isolated it to elucidate its structure. The isolated alkaloid proved to be a mixture of two stereoisomers. The structures of the two compounds as determined by 1D and 2D NMR spectroscopy, were E-thesinine-O-4'-alpha-rhamnoside (1) and Z-thesinine-O-4'-alpha-rhamnoside (2). These identifications were supported by the characterisation by GC-MS and optical rotation of (+)-isoretronecanol as the necine base released on alkaline hydrolysis of these alkaloids. 1 and 2 together with the aglycone and a hexoside were also detected in tall fescue (Festuca arundinacea). This is the first report of pyrrolizidine alkaloids produced by grasses (Poaceae).     

Choline esterase inhibitory properties of alkaloids from two Nigerian Crinum species

The bulbs of Crinum jagus and Crinum glaucum are used in traditional medicine in southern Nigeria for memory loss and other mental symptoms associated with ageing. Alkaloidal extracts of bulbs from each species showed inhibition of acetylcholinesterase, an activity exploited therapeutically to raise the depressed levels of acetylcholine in the brain associated with Alzheimer's disease. Using the in situ bioautographic test method for enzyme inhibition, a number of alkaloids were isolated and their activity quantified using the Ellman spectrophotometric test. The most active alkaloids isolated were hamayne (IC50 250 microM) and lycorine (IC50 450 microM) whilst other alkaloids were comparatively inactive with haemanthamane giving 3% inhibition and crinamine giving 4.4% inhibition at 50 mg ml(-1) (174 microM). These contrast with the positive control physostigmine which gave IC50 of 0.25 microM. Cholinesterase activity appears to be associated with the presence of two free hydroxy groups in this structural type of Amaryllidaceae alkaloid.     

Norditerpenoid alkaloids from Delphinium species

From the aerial parts of four Delphinium species 11 known and 3 new norditerpenoid alkaloids have been isolated: from D. dissectum Huth: delavaine A/B, deoxylycoctonine, methyllycaconitine; new: 10-hydroxymethyllycaconitine; from D. excelsum Reichenb.: delcaroline, delectinine, delterine, methyllycaconitine; new: 10-hydroxymethyllycaconitine, 18-O-methyldelterine and 10-hydroxynudicaulidine; from D. grandiflorum L.: delcosine, deltatsine, grandiflorine, methyllycaconitine; from D. triste Fisch.: delcosine, macrocentridine, 14-dehydrodelcosine. The structures of the new alkaloids were established on the basis of MS, 1H, 13C, DEPT, homonuclear COSY, HMQC and HMBC NMR spectroscopic techniques.     

Secondary and tertiary isoquinoline alkaloids from Xylopia parviflora

From the secondary and tertiary alkaloidal fractions of the root and the bark of Xylopia parviflora (Annonaceae), the isoquinoline alkaloids, 10,11-dihydroxy-1,2-dimethoxynoraporphine and parvinine were isolated, along with 39 known alkaloids. Their structures were determined on the basis of analysis of spectroscopic data.     

Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants

Camptothecin derivatives are clinically used anti-neoplastic alkaloids that biogenetically belong to monoterpenoid indole alkaloids. Camptothecin-related alkaloids from the methanol extracts of Ophiorrhiza pumila, Camptotheca acuminata and Nothapodytes foetida plants were profiled and identified using a reverse-phase high performance liquid chromatography coupled with on-line photodiode array detection and electrospray-ionization ion-trap mass spectrometry. A natural 10-glycosyloxy camptothecin, chaboside, was accumulated in tissues of O. pumila but not in C. acuminata and N. foetida. Anthraquinones regarded as phytoalexins were present in the extracts of hairy roots and calli but not in the differentiated plants of O. pumila. These findings demonstrated a remarkable difference in the constituents between the differentiated plants and the hairy roots or calli tissues. The activity of strictosidine synthase, a key enzyme of camptothecin biosynthesis, was detected in the protein extracts of stems and roots of O. pumila, being correlated with the pattern of strictosidine synthase mRNA expression.     

Insecticidal sesquiterpene pyridine alkaloids from Euonymus species

Three insecticidal sesquiterpene pyridine alkaloids with a beta-dihydroagarofuran sesquiterpene skeleton, euoverrine A (1), B (2), and euophelline (3), and a known compound, euojaponine C (4), were isolated from the root bark of Euonymus verrucosides, E. fortunei and E. phellomana by bioassay-guided fractionation. Their chemical structures were elucidated mainly by analyses NMR and MS spectral data.     

Norditerpene and diterpene alkaloids from Aconitum variegatum

Aerial parts of Aconitum variegatum L. from the Pyrenees furnished four norditerpene alkaloids, 16 beta-hydroxycardiopetaline, 8-ethoxysachaconitine, 14-acetylgenicunine B, N-deethyl-N-19-didehydrosachaconitine, five diterpene alkaloids 15-veratroyldictizine, 15-veratroyl-17-acetyldictizine, 15-veratroyl-17-acetyl-19-oxodictizine, N-ethyl-1 alpha-hydroxy-17-veratroyldictizine, variegatine and the known alkaloids sachaconitine, 14-O-acetylsachaconitine, karakoline, talatizamine, 10-hydroxytalatizamine, 14-acetyltalatizamine, 14-acetyl-10-hydroxytalatizamine, N-methylarmepavine, pengshenin B, delsoline, dihydrodelsoline, delcosine and genicunin B. Structures of the alkaloids were established by MS, 1D- and 2D-NMR techniques.     

Cyclic peptide alkaloids from the bark of Discaria americana

The isolation and structure determination of cyclic peptide alkaloids, discarine-M and discarine-N, along with seven known cyclic peptide alkaloids, adouetine-Y', franganine, frangulanine, discarines-A, -B, -C, and -D from the root bark of Discaria americana are described. Structures were determined spectroscopically, especially using 2D NMR spectroscopic analysis. The crude methanol extract, the basic ether extract, and the alkaloids 6 and 7 also weakly inhibited growth of gram-negative and gram-positive bacteria.     

Heteroyohimbinoid type oxindole alkaloids from Mitragyna parvifolia

The leaves of the plant Mitragyna parvifolia have afforded two alkaloids, 16,17-dihydro-17beta-hydroxy isomitraphylline (1) and 16, 17-dihydro-17beta-hydroxy mitraphylline (2), together with two known alkaloids, isomitraphylline (3) and mitraphylline (4). The structures of 1 and 2 were elucidated using 1D and 2D NMR spectral methods, including (1)H-(1)HCOSY, DEPT, HSQC, and HMBC. Mitraphylline was the main alkaloid constituent.     

Brassicaceae contain nortropane alkaloids

The report of cochlearine, the 3-hydroxybenzoate ester of tropine found in Cochlearia officinalis, Brassicaceae, initiated a screening for tropane alkaloids in Cochlearia species and for calystegines in further Brassicaceae. All ten Cochlearia species investigated contained cochlearine, tropine, and pseudotropine. Calystegines, nortropane alkaloids deriving from pseudotropine, were also identified in all Cochlearia species and accumulated up to 0.5% dry mass in leaves. Brassicaceae species of all major lineages of the family were analysed for calystegines. Of the 43 species included in the study, 18 accumulated calystegines of various structures. This is the first screening of Brassicaceae for products of the tropane alkaloid pathway, which is known as characteristic for plants of Solanaceae family. The identification of calystegines in all branches of the Brassicaceae family including Aethionema, a species at the basis of the family, suggests tropane alkaloids as secondary compound typical for Brassicaceae.     

Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.     

Dechlorodauricumine from cultured roots of Menispermum dauricum

Dechlorodauricumine, a possible organic substrate for biochlorination, was isolated from cultured roots of Menispermum dauricum, a rich source of chlorinated alkaloids. Its structure was established by spectroscopic and chemical methods.     

Intraspecific variability in the alkaloid metabolism of Galanthus elwesii

Alkaloid pattern of individuals from 16 Bulgarian Galanthus elwesii populations was investigated by GC/MS and TLC. Twenty-one Amaryllidaceae alkaloids were detected and 14 of them were identified. Crinane type alkaloids, haemanthamine or crinine, dominated alkaloid metabolism in most of the populations. With exception of one population, where the separate individuals showed variable alkaloid profiles (dominated by crinine or haemanthamine) the individuals of the rest of populations have identical and characteristic alkaloid profiles. Some populations showed remarkable differences in respect to their alkaloid pattern-type of biosynthesis, main alkaloids and number of alkaloids. Populations dominated by galanthamine type alkaloids were found as well. These data demonstrate that like the morphological features, the alkaloid metabolism of G. elwesii is also variable.     

The role of chemical fingerprinting: application to Ephedra

Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in Traditional Chinese Medicine (TCM). Preparations, namely teas, of E. sinica have been used for over 5000 years as a stimulant and as an antiasthmatic. In the West, extracts of E. sinica, E. intermedia or E. equisetina are most commonly used in dietary supplements as a stimulant and to promote weight loss. More than 50 species of Ephedra are native to both hemispheres, but the detection of ephedrine alkaloids has been limited to species in Eurasia. Currently, methods exist to quantitate the ephedrine alkaloids in extracts of plant material or dietary supplements, but the methods are not able to verify the extract is of an Ephedra species. Reverse phase high performance liquid chromatography with photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two regions of comparison were determined in the chromatograms at 320 nm. The series of peaks between 52 and 64 min confirms an Ephedra species is being analyzed. The aforementioned peaks also could distinguish between Ephedra species from Eurasia, North America and South America. Peaks at ca. 57 and 59 min were isolated and determined to be two new compounds, 4-(2-eicosyloxycarbonyl-vinyl)-benzoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid respectively. Authentication of ground plant material as Ephedra can be achieved by this chemical fingerprinting method.