Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission.
The leaves of Catharanthus roseus constitute the only source of the well known indolomonoterpenic alkaloids vincristine and vinblastine. In this work we studied the biological potential of the roots, which are used in several countries as decocts or hot water extracts for the treatment of a number of conditions. The aqueous extract strongly inhibited acetylcholinesterase (AchE) in an in vitro microassay, an effect ascribable mainly to serpentine ([IC.sub.50] = 0.775 [micro]M vs physostigmine [IC.sub.50] = 6.45 [micro]M) as assessed with the pure compound. Pure alkaloids were tested for muscarinic and nicotinic antagonism using rat ex-vivo preparations, namely, ileum and diaphragm/phrenic-nerve, respectively. Serpentine competitively blocked muscarinic receptors with a [pA.sub.2] of 5.2, whereas the precursor ajmalicine up to 80 [micro]M was undistinguishable from control, and catharanthine exhibited an unsurmountable muscarinic antagonism at greater than 10 [micro]M concentrations. Nicotinic receptor mediated diaphragm contractions were fully inhibited by catharanthine ([IC.sub.50] = 59.6 [micro]M) and ajmalicine ([IC.sub.50] = 72.3 [micro]M), in a reversible but non-competitive manner, unlike the more potent nicotinic antagonist tubocurarine ([IC.sub.50] = 0.35 [micro]M) whose competitive blockade was overcome by a physostigmine-induced increase in acetylcholine. Serpentine up to 100 [micro]M did not change diaphragm contractions suggesting reduced affinity for neuromuscular nicotinic receptors. Despite strong in vitro AchE inhibition, serpentine failed to restore diaphragm contractions upon submaximal tubocurarine blockade, suggesting that poor tissue penetration may prevent serpentine from inhibiting AchE in deep neuromuscular synapses in the ex-vivo preparation. To our knowledge, the present study is the first to assess the effect of C. roseus root extracts, as well as of serpentine, ajmalicine and catharanthine on AchE. The results described herein suggest that the currently overlooked C. roseus roots may constitute a promising source of compounds with pharmaceutical interest. Moreover, given serpentine's potent in vitro AchE inhibitory activity and low cholinergic receptor affinity, it is conceivable that minor structural modifications may yield a potent and selective AchE inhibitor, potentially useful for the pharmacological management of conditions such as Alzheimer's disease and/or myasthenia gravis.
Keywords: Catharanthus roseus roots Catharanthine Ajmalicine Serpentine Acetylcholinesterase Cholinergic Receptors
In the area of natural products, Catharanthus roseus is an unavoidable species. Since the 50's that the anticancer activity of some of its indolomonoterpenic alkaloids, vincristine and vinblastine, is known, and they have been used extensively in several malignant conditions, such as Hodgkin's and non-Hodgkins's lymphomas, acute lymphoblastic leukaemia, neuroblastoma, breast carcinoma, among others (Dong et al. 1995). For biosynthetic reasons (Fig. 1), the condensation of catharanthine and vindoline is an absolute requirement for the formation of vinblastin and, later, vincristine (Greenblatt et al. 2004). However, the fact that vindoline does not exist in the roots of C. roseus, but only in the green parts of the plant, renders that no vincristine nor vinblastin can be found in the roots of this species. Other molecules with important biological activity can be obtained from C. roseus, namely ajmalicine, used as anti-hypertensive, and serpentine, with sedative activity (Sottomayor and Ros Barcelo 2005; Van der Heijden et al. 2004). In fact, C. roseus revealed to be a remarkable factory of bioactive compounds and over 130 compounds have already been described to these days. C. roseus is the leading single plant species reported to produce such a wide array of complex alkaloids (Blasko and Cordell 1990).
[FIGURE 1 OMITTED]
Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system that is characterized by profound memory impairment, emotional disturbance, and, in late stages, by personality changes (Bartolucci et al. 2001). Neurochemical and neuroanatomical studies suggest that cholinergic neurons projecting to the neocortex and hippocampus are those predominantly affected in AD. This led to the cholinergic hypothesis, which associates AD symptoms to cholinergic deficiency. Therefore, symptomatic treatment for AD has been focused upon augmenting brain cholinergic neurotransmission. An effective way to increase acetylcholine levels is to inhibit acetylcholinesterase (AchE), the enzyme responsible for degrading acetylcholine (Ach) in the synaptic cleft (Greenblatt et al. 2004).
Within AchE inhibitors, the natural alkaloids huperzine A and galanthamine, as well as the synthetic compounds rivastigmine (an analogue of physostigmine, an excellent inhibitor of AchE but suffering from poor intestinal absorption in humans) and tacrine are used. Donepezil is a piperidine-type cholinesterase inhibitor, which exerts reversible, non-competitive inhibition of acetylcholinesterase, being structurally distinct from the other cholinesterase inhibitors available today.
Among natural products, alkaloids seem to be the most promising candidates for AchE inhibitors, given their complex and nitrogen-containing structure. Several compounds, with distinct structures, have been reported, including steroidal alkaloids from Amaryllidaceae (Lopez et al. 2002) and Buxaceae (Rahman and Choudhary 2001), isoquinolines from Papaveraceae (Kim 2002; Kim et al. 2004), quinolizidines from Lycopodiaceae (Orhan et al. 2004), glycoalkaloids from Solanaceae (Roddick 1989) and indole alkaloids from Leguminosae (Karczmar 1998). In what concerns to Apocynaceae, in which C. roseus is included, indole alkaloids with AchE inhibitory activity were described only in Tabernaemontana australis (Andrade et al. 2005).
Every year, about 1000 tons of C roseus leaves are used for the extraction of the alkaloids with medicinal use (vincristine and vinblastine), leaving tons of unused plant material (Pelt 2001). The present study explored C. roseus roots as a source of lead compounds for the pharmaceutical industry, namely AchE inhibitors, thus making use of all the amounts of roots that arise from the industrial exploitation of the leaves for the extraction of the anticancer alkaloids.
Materials and methods
Standards and reagents
Acetylcholinesterase, acetylthiocholine iodide (ATCI), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), carbachol, d-tubocurarine, physostigmine, serpentine and ajmalicine were from Sigma-Aldrich (St. Louis, MO, USA). Catharanthine sulfate was from Gedeon Richter Ltd (Budapest, Hungary). Water was treated in a Milli-Q (Millipore, Bedford, Massachusetts) water purification system.
Plants of Catharanthus roseus (L.) G. Don cv. Little Bright Eye were grown at 25[degrees]C in a growth chamber, under a 16 h photopehod, using white fluorescent light at a photon flux density of 70 [micro]mol/[m.sup.2]/s. Roots from three distinct plants were collected, frozen (-20[degrees]C) and lyophilized. Dried material was powdered (910 [micro]m) and kept in a desiccator at room temperature, in the dark, until their analysis. Voucher specimens were deposited at the Department of Pharmacognosy of the Faculty of Pharmacy of Porto University (CATROS-R0109).
For the biological assays performed, an aqueous extract was prepared: 1.5 g of lyophilized material were extracted with 300 ml of boiling water for 20 minutes, with subsequent filtration. The resulting extract was then lyophilized and maintained in a desiccator at room temperature, in the dark.
In vitro acetylcholinesterase inhibitory activity
The following buffers were used. Buffer A: 50 mM Tris-HCl, pH 8; buffer B: 50 mM Tris-HCl, pH 8, containing 0.1% bovine serum albumin (BSA); buffer C: 50 mM Tris-HCl, pH 8, containing 0.1 M NaCl and 0.02 M MgC1.6[H.sub.2]O.
Acetylcholinesterase was from electric eel (type VI-s, lyophilized powder, 425 U/mg, 687 mg/protein). Lyophilized enzyme was dissolved in buffer A to make 1000 U/ml stock solution, and further diluted with buffer B to get 0.44 U/ml enzyme for the microplate assay.
We followed the method described by Rhee and colleagues (Rhee et al. 2001), with modifications (Pereira et al. 2009). AchE activity was measured using a 96-well Multiskan Ascent microplate reader (Thermo, Electron Corporation) based on Ellman's method. The substrate acetylthiocholine is hydrolyzed by the enzyme to thiocholine, which reacts with Ellman's reagent (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate, detected at 405 nm. In each well the mixture consisted of 25 [micro]l of 15 mM ATCI in water, 125 [micro]l of 3 mM DTNB in buffer C, 50 [micro]l of buffer Band 25 [micro]l of sample dissolved in a solution of 10% methanol in buffer C and the absorbance was measured at 405 nm every 13 s for five times. After this step 25 [micro]l of 0.44 U/ml of enzyme was added and the absorbance was read again every 13 s for eight times. The rates of reactions were calculated by Ascent Software version 2.6 (Thermo Labsystems Oy). The rate of the reaction before adding the enzyme was subtracted from that obtained after adding the enzyme in order to correct eventual spontaneous hydrolysis of substrate. Percentage of inhibition was calculated by comparing the rates of the sample with the control (10% methanol in buffer C). Catharanthine, serpentine and ajmalicine were individually tested, as well as in mixture, in representation of the main compounds.
Ex vivo cholinergic antagonism and acetylcholinesterase inhibition
Male Wistar rats (300 - 350 g; Charles River, Barcelona, Spain) were used to obtain small intestine (ileum) fragments, for testing muscarinic receptor antagonism, and diaphragm/phrenic-nerve preparations, for testing nicotinic receptor antagonism and acetylcholinesterase inhibition. Animal handling and care followed the EU guidelines (86/609/EEC) and Portuguese law (1005/92 and 1131/97). Biological preparations were mounted under a resting tension of 1 g in organ baths [20 ml volume for ileum segments (ca. 2 cm), and 40 ml volume for diaphragm/phrenicnerve preparations] containing Krebs solution (NaCl 120.0 mM, KC1 5.0 mM, [CaCl.sub.2] 2.5 mM, [MgSO.sub.4] 1.0 mM, [NaHCO.sub.3] 25.0 mM, and glucose 10 mM, pH 7.4). Experiments were performed at 37[degrees]C with continuous aeration with carbogen (95% [O.sub.2] and 5% [CO.sub.2]), with regular renovation of the Krebs solution. Muscle contractions (ileum longitudinal muscle, or diaphragm) were measured with isotonic transducers and recorded in chart polygraphs (Ugo Basile, Italy).
Muscarinic receptor antagonism
Individual alkaloids, ajmalicine, catharantine, and serpentine were tested for muscarinic receptor antagonism in the rat ileum preparation. Following ileum stabilization, cumulative concentration response curves to the muscarinic agonist carbachol were performed in the absence and in the presence of increasing concentrations of the individual alkaloids. Non-linear regression with GraphPad Prism (v. 5.0 GraphPad Software, CA, USA) was used to analyze concentration-response curves. Antagonism with parallel curve displacements without decreases in maximal response was analyzed by Schild Regression.
Nicotinic receptor antagonism and acetylcholinesterase inhibition
Individual alkaloids were tested for their ability to block nicotinic receptor dependent contractions of the rat diaphragm/phrenic-nerve, in comparison with the neuromuscular nicotinic antagonist tubocurarine. Regular twitch contractions of the diaphragm were induced by electric stimulation of the phrenic nerve (0.2 Hz, 1 ms, SV) via platinum electrodes and a square wave stimulator. After stabilization (20min), tubocurarine or individual alkaloids were added cumulatively in order to establish concentration-inhibition curves. The type of antagonism was inferred in independent preparations by testing the ability of the acetylcholinesterase inhibitor physostigmine to reverse the inhibition of contractions, namely by increasing acetylcholine concentration in the synapses and displacing the antagonist (reversible by physostigmine = competitive blockade; non-reversible by physostigmine = non-competitive blockade). Restoration of contractions upon washing out the drug (by renewing the organ bath Krebs solution) was taken as an indication of reversible binding of the alkaloid. To test for the acetylcholinesterase inhibitory activity in these preparations, a submaximal (about 80%) inhibition of contractions was induced by titrating tubocurarine, and then adding the alkaloid and assessing whether contractions increased, using physostigmine as a positive control.
Results and discussion
Binding to the AchE enzyme takes place by means of bonds established with its histidine, serine and glutamic acid residues, normally through different chemical groups that usually require at least a tertiary nitrogen atom. For this fact, among natural products alkaloids are usually the standard inhibitors, including physostigmine and galanthamine (Greenblatt et al. 2004). Several reviews are available concerning cholinesterase inhibitors from natural sources, with the majority of inhibitors being alkaloids (Loizzo et al. 2008; Hostettmann et al. 2006; Orhan et al. 2009).
AchE inhibitory activity was reported before in C. roseus leaves, stems, and petals (Pereira et al. 2009). However no alkaloid was determined in that work. As stated before, some alkaloids meet the perfect requirements for AchE inhibitors, making their analysis very important. The composition of the aqueous extract of C. roseus roots has recently been reported by our group (Ferreres et al. 2009) and several alkaloids were determined: 19-S-vindolinine (ca. 13% of total identified alkaloids), vindolinine (ca. 11%), ajmalicine (ca. 4%) and an ajmalicine isomer (not quantified), tabersonine (not quantified), catharanthine (ca. 8%), serpentine (ca. 46%) and a serpentine isomer (ca. 18%).
C. roseus roots' aqueous extract displayed a concentration-dependent inhibition of the enzyme (Fig. 2A), reaching nearly 100% of inhibition under the tested concentrations. The calculated [IC.sub.50] was 25.5 [micro]g/ml (Table 1).
Table 1 Comparison of AchE inhibitory activity between Catharanthus roseus roots, other plant parts and several alkaloids. Values represent [IC.sub.50] ([micro]g/ml). C. roseus material Alkaloid Leaves * Stems * Seeds * Petals * Roots S (a) S+A+C (a) 422 441.8 - 2683.1 25.5 0.273 2.249 (a) S, serpentine; A. ajmalicine; C, catharantine. * From Pereira et al. 2009.
The activity observed herein with the roots (Table 1) was much higher than that found in any plant part available in literature: leaves ([IC.sub.50] 422.0 [micro]g/ml), stems ([IC.sub.50] 441.8 [micro]g/ml) and petals ([IC.sub.50] 2683.1 [micro]g/ml) (Pereira et al. 2009). These results represent a 17 to 110 fold higher potency. In addition, almost 100% of inhibition could be achieved using roots' extract, against about 85% with other plant parts.
[FIGURE 2 OMITTED]
To assess the role of the identified alkaloids in the displayed activity, several experiments were conducted using pure standards in the amounts quantified in the extract (Ferreres et al. 2009), both individually and in combination. At the concentrations occurring in the extract, the mixture of serpentine, plus ajmalicine and catharanthine revealed a concentration-dependent inhibitory effect, with an [IC.sub.50] at ca. 2.25 [micro]g/ml. However. when tested isolated, serpentine (Fig. 2B), the major compound found in the extract, displayed an [IC.sub.50] of ca. 0.27 [micro]g/ml (0.775 [micro]M), a surprisingly low value. This compound alone accounted for an [IC.sub.50] almost a hundred times lower than that of the crude extract. The potency of this compound led us to apply the same assay on a well established acetylcholinesterase inhibitor, physostigmine (Fig. 2B), with an [IC.sub.50] of 1.77 [micro]g/ml (6.45 [micro]M) being found. Thus, serpentine was nearly 10 times more potent than physostigmine in this in vitro assay. When ajmalicine or catharanthine were assayed no inhibitory effect on the enzyme was found, which is in accordance with the higher [IC.sub.50] value obtained for their mixture with serpentine.
A closer look to serpentine's structure may provide some leads for its potent inhibitory activity, In the structure of AchE enzyme an anionic binding region exists. As so, the quaternary nitrogen that serpentine bears could be able to bound to the negatively charged aspartate residue by means of ionic interactions. In addition, serpentine may interact with aromatic amino acids by induced dipole interactions (Patrick 2005).
Acetylcholinesterase inhibition is potentially useful in symptomatic control of Alzheimer's disease (CNS action) or myasthenia gravis (muscular action). Also, blockade of cholinergic receptors may aggravate AD symptoms, and prevent the increase in cholinergic transmission expected from increased acetylcholine in synapses as result of cholinesterase inhibition. Therefore, we assessed the ability of the individual alkaloids to antagonize cholinergic receptors (both muscarinic and nicotinic). Moreover, we tested whether the potent in vitro inhibition of acetycholinesterase by serpentine was also exhibited in a more physiological ex vivo neuro-muscular preparation, namely the diaphragm/phrenic-nerve preparation.
Individual alkaloids, ajmalicine, catharantine, and serpentine, were tested for effects as muscarinic antagonists. Cumulative concentration-response curves to carbachol were performed in the rat ileum preparation, in the absence or presence of increasing concentrations of the alkaloids. Ajmalicine up to 80 [micro]M was undistinguishable from control (Fig. 3B vs. 3A), excluding any significant muscarinic blockade. Catharanthine evoked a concentration-dependent attenuation of carbachol responses, producing rightward curve displacements and decreases in maximal agonist responses, thus showing unsurmountable (noncompetitive) antagonistic activity (Fig. 3C). Serpentine induced concentration-dependent parallel displacements of carbachol curves without decrease in maximal response (Fig. 3D). Schild regression analysis of serpentine data yielded a slope of 1.14 [+ or -] 0.05, and a p[A.sub.2] of 5.24 [+ or -] 0.29, suggesting a reversible competitive antagonism with a dissociation equilibrium constant ([K.sub.B]) of about 6.5 [+ or -] 3.5 [micro]M (n=3 independent measurements in different rats).
Nicotinic receptor antagonism and ex-vivo acetylcholinesterase inhibition
Individual alkaloids, ajmalicine, catharantine, and serpentine, were tested for their ability to antagonize nicotinic receptors at the neuromuscular junction. Diaphragm contractions induced by phrenic nerve stimulation were fully inhibited by tubocurarine in a concentration-dependent manner ([IC.sub.50]=0.35 [+ or -] 0.05 [micro]M, n=4), thus showing their dependence on nicotinic receptor activation (Fig. 4A). Catharantine and ajmalicine also inhibited contractions in a concentration-dependent manner but were considerably less potent than tubocurarine ([IC.sub.50]=59.6 [+ or -]7.1 [micro]M, and 72.3 [+ or -] 22.5 [micro]M, n=3, for catharanthine and ajmalicine, respectively), whereas serpentine up to 100 [micro]M exhibited no antagonistic activity on diphragm contractions (Fig. 4B). As expected, tubocurarine inhibition was reversible by acetylcholinesterase inhibition with physostigmine (9 [micro]M; Fig. 4C) revealing its competitive interaction, as it is displaced by increased acetylcholine. The inhibition of contractions by either catharanthine or ajmalicine was not reversibie by physostigmine, being restored after washout of the alkaloids (not shown), thus suggesting a non-competitive (unsurmountable) but reversible form of nicotinic antagonism.
[FIGURE 3 OMITTED]
Given that serpentine exhibited a potent acetylcholinesterase inhibitory activity in the in vitro assay with the isolated enzyme, we tested whether serpentine would reverse tubocurarine blockade of diaphragm contractions as occurs for physostigmine (Fig. 4C). As shown in Fig. 4D, representative of 3 independent experiments, serpentine up to 42 [micro]M did not increase contractions of the diaphragm, even though subsequent application of physostigmine was able to do so. This was unexpected given the reported agreement between in vitro electric eel acetylcholinesterase inhibition and in vivo activity in the rat (Snape et al. 1999). A possible explanation is that while serpentine inhibits isolated acetylcholinesterase, it may fail to reach acetylcholinesterase deep in the cholinergic synapses at the neuromuscular junction due to poor tissue penetration. Alternatively, despite high homology, eel cholinesterase may differ from that in the rat at a critical serpentine interaction site, being presently unknown whether it would affect the human enzyme. Nevertheless, considering that serpentine's has: (i) high in vitro potency (IC50 = 0.775 [micro]M) as acetylcholinesterase inhibitor in comparison with physostigmine (6.45 [micro]M); (ii) a competitive and low affinity interaction with muscarinic receptors ([K.sub.b] = 6.5 [micro]M); and (iii) no neuromuscular nicotinic antagonism up to 100 [micro]M, serpentine may be an interesting lead compound. Indeed, minor structural modifications to serpentine may yield a potent and selective cholinesterase inhibitor, potentially useful for pharmacological management of Alzheimer's disease and/or myasthenia gravis, thus highlighting the interest of C. roseus roots as a source of compounds with pharmaceutical interest. For this purpose, further studies addressing the question of this compound's toxicity, bioavailability and pharmacokinetics should be conducted, as the only data available is the lethal dose of serpentine and related compounds in frogs, which corresponded to 0.5 mg per kilo of frog, being the value found for rats four times higher (Chopra 1958). The pharmacological action of this drug on higher animals, such as cats, was also tested (Chopra 1958). The authors found that water extracts containing serpentine injected intravenously in animals produced no appreciable effects. However, some caution must be taken when interpreting these results, as the techniques used in the referred work could have been inappropriate.
[FIGURE 4 OMITTED]
The authors are grateful to Fundacao para a Ciencia e a Tecnologia (FCT) for financial support (PTDC/AGR-AAM/64150/2006). J. Faria (BII) is grateful to FCT for the grant. The authors also wish to thank Maria Fernanda Pereira and Maria do Ceu Pereira for able technical assistance.
Andrade, M.T., Lima, J.A., Pinto, A.C., Rezende, C.M., Carvalho, M.P., Epifanio, R.A., 2005. Indole alkaloids from Tabemaemontana australis (Muell. Arg) Miers that inhibit acetylcholinesterase enzyme. Bioorg. Med. Chem. 13, 4092-4095.
Bartotucci, C., Perola, E., Pilger, C., Fels, G., Lambal, D., 2001. Three-dimensional structure of a complex of galanthamine (Nivalin R ) with acetylcholinesterase from Torpedo californica: implications for the design of new anti-Alzheimer drugs. Proteins 42, 182-191.
Blasko, G., Cordell, G.A., 1990. Isolation, structure elucidation, and biosynthesis of the bisindole alkaloids of Catharanthus. In: Brossi, A., Suffness, M. (Eds.), The Alkaloids. Academic Press, pp. 1-76.
Chopra, R.N., 1958. In: Chopra's indigenous drugs of India. Academic Publishers, Kolkata.
Dong, J.G., Bornmann, W., Nakanishi, K., Berova, N., 1995. Structural studies of vinblastine alkaloids by exciton coupled circular dichroism. Phytochemistry 40, 1821-1824.
Ferreres, F., Pereira, D.M., Valentao, P., Oliveira, J.M.A., Faria, J., Caspar, L., Sottomayor, M., Andrade, P.B., 2009. Simple and reproducible HPLC-DAD-ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots. J. Pharm. Biomed. Anal. 51, 65-69.
Greenblatt, H.M., Guillou, C., Guenard, D., Argaman, A., Botti, S., Badet, B., Thal, C., Silman, I., Sussman, J.L., 2004. The complex of a bivalent derivative of galanthamine with Torpedo acetylcholinesterase displays drastic deformation of the active-site gorge: implications for structure-based drug design. J. Am. Chem. Soc. 126, 15405-15411.
Hostettmann, K., Borloz, A., Urbain, A., Marston, A., 2006. Natural product inhibitors of acetylcholinesterase. Curr. Org. Chem. 10, 825-847.
Karczmar, A., 1998. Anticholinesterases: dramatic aspects of their use and misuse. Neurochem. Int. 32, 401-411.
Kim, D.K., 2002. Inhibitory effect of corynoline isolated from the aerial parts of Corydalis incisa on the acetylcholinesterase. Arch. Pharm. Res. 25, 817-819.
Kim, D.K., Lee, K.T., Baek, N.I., Kim, S.H., Park, H.W., Lim, J.P., Shin, T.Y., Eom, D.O., Yang, J.H., Eun, J.S., 2004. Acetylcholinesterase inhibitors from the aerial parts of Corydalis speciosa. Arch. Pharm. Res. 27, 1127-1131.
Loizzo, M.R., Tundis, R., Menichini, F., Menichini, F., 2008. Natural products and their derivatives as cholinesterase inhibitors in the treatment of neurodegenerative disorders: an update. Curr. Med. Chem. 15, 1209-1228.
Lopez, S., Bastida. J., Viladomat, F., Codina. C., 2002. Acetylcholinesterase inhibitory activity of some Amaryllidaceae alkaloids and Narcissus extracts. Life Sci. 71, 2521-2529.
Orhan, I., Sener, B., Choudhary, M.I. Khalid, A., 2004. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants. J. Ethnopharmacol. 91, 57-60.
Orhan, G., Orhan, I., Oztekin-Subutay, N., Ak, F., Sener, B., 2009. Contemporary anticholinesterase pharmaceuticals of natural origin and their synthetic analogues for the treatment of Alzheimer's disease. Recent Pat. CNS Drug Discov. 4, 43-51.
Patrick, G.L, 2005. In: An Introduction to Medicinal Chemistry 3rd ed. Oxford Press, pp. 581-583.
Pelt, J.M., 2001. In: Les nouveaux remedes naturels. Editions Fayard, Paris.
Pereira, D.M., Ferreres, F., Oliveira, J., Valentao, P., Andrade, P.B., Sottomayor, M., 2009, Targeted metabolite analysis of Catharanthus roseus and its biological potential. Food Chem. Toxicol. 47, 1349-1354.
Rahman, A.U., Choudhary, M.I., 2001. Bioactive natural products as a potential source of new pharmacophores. A theory of memory. Pure Appl. Chem. 73, 555-560.
Rhee, K., van de Meent, M., Ingkaninan, K., Verpoorte, R., 2001. Screening for acetylcholinesterase inhibitors from Amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining. J. Chromatogr. A 915, 217-223.
Roddick, J.G., 1989. The acetylcholinesterase-inhibitory activity of steroidal glycoalkaloids and their aglycones. Phytochemistry 28, 2631-2634.
Snape, M.F., Misra, A., Murray, T.K., De Souza, R.J., Williams, J.L., Cross, A.J., Green, A.R., 1999. A comparative study in rats of the in vitro and in vivo pharmacology of the acetylcholinesterase inhibitors tacrine, donepezil and NXX-066. Neuropharmacology 38, 181-193.
Sottomayor, M., Ros Barcelo, A., 2005. The Vinca alkaloids: from biosynthesis and accumulation in plant cells, to uptake, activity and metabolism in animal cellsIn: Atta-ur-Rahman (Ed.). Studies in Natural Products Chemistry (Bioactive Natural Products). Elsevier Science Publishers, The Netherlands, pp. 813-857.
Van der Heijden, R., Jacobs, D.I., Snoeijer, W., Hallard, D., Verpoorte, R., 2004. The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr. Med. Chem. 11, 607-628.
David M. Pereira (a), Federico Ferreres (b), Jorge M.A. Oliveira (c), Luis Gaspar (a), Joana Faria (a), Patricia Valentao (a), Mariana Sottomayor (d), *, Paula B. Andrade (a), *
(a) REQUIMTE/Department of Pharmacognosy, Faculty of Pharmacy, Porto University, R. Anibal Cunha, 164, 4050-047 Porto, Portugal
(b) Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University Espinardo (Murcia), Spain
(c) REQUIMTE, Servico de Farmacologia, Faculdade de Farmacia, Universidade do Porto, R. Anibal Cunha, 164, 4050-047 Porto, Portugal
(d) IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto and Departamento de Botanica, Faculdade de Ciencias, Universidade do Porto, R. Campo Alegre 823, 4150-180 Porto, Portugal
* Corresponding authors: Tel.: +351 222078935; fax: +351 222003977.
E-mail addresses: email@example.com (M. Sottomayor). firstname.lastname@example.org (P.B. Andrade).
0944-7113/$ - see front matter [C] 2009 Elsevier GmbH. All rights reserved.
|Printer friendly Cite/link Email Feedback|
|Author:||Pereira, David M.; Ferreres, Federico; Oliveira, Jorge M.A.; Gaspar, Luis; Faria, Joana; Valentao, P|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jul 1, 2010|
|Previous Article:||The pharmacokinetics of C-glycosyl flavones of Hawthorn leaf flavonoids in rat after single dose oral administration.|
|Next Article:||An aqueous extract of Ammi visnaga fruits and its constituents khellin and visnagin prevent cell damage caused by oxalate in renal epithelial cells.|