Enzymatic Inhibition Studies of Selected Flavonoids and Chemosystematic Significance of Polymethoxylated Flavonoids and Quinoline Alkaloids in Neoraputia ( Rutaceae )

Nosso interesse quimiotaxonômico sobre Neoraputia nos estimulou a examinar N. paraensis, visando a busca de alcalóides. As frações foram monitoradas via RMN H e ESI-EM/EM e foram analisadas somente aquelas cujos espectros apresentavam características de alcalóides do ácido antranílico e flavonóides não isolados anteriormente. Foram isolados do caule os alcalóides flindersina, skimmianina, 8-metoxiflindersina e dictamnina; das folhas os flavonóides 3',4',7,8-tetrametoxi-5,6(2",2"-dimetilpirano)-flavona, 3',4',5,7,8-pentametoxiflavona, 5-hidroxi-3',4',6,7-tetrametoxiflavona, 3',4'-metilenodioxi-5,6,7-trimetoxiflavona e 5-hidroxi-3',4'-metilenodioxi-6,7-dimetoxiflavona,. Os alcalóides do ácido antranílico não foram encontrados em dez anos. Vários flavonóides isolados de N. paraensis, N. magnifica, Murraya paniculata, enxerto de Citrus sinensis (Rutaceae) e Lonchocarpus montanus (Leguminosae) foram testados frente a gliceraldeído-3-fosfato desidrogenase de Trypanosoma cruzi, visando verificar seus potenciais em inibir a atividade da enzima. Os flavonóides polimetoxilados e um isoflavonóide foram os mais ativos.


Introduction
Neoraputia species were originally described by Engler as species of Raputia Aublet.Emmerich later proposed the new genus Neoraputia Emmerich to accommodate six species, four of them from Raputia and two new ones.These two genera are assigned to the tribe Cusparieae. 1 Previous investigations of Neoraputia reported the presence of eleven polymethoxylated flavonoids, six flavones, three 5,6-(2",2"dimethylpyrano)-flavones, one 6,7-(2",2"-dimethylpyrano)flavone and one flavanone from N. alba (Engler) Emmerich; 2,3 five polymethoxylated flavones and two flavanones, 2'hydroxy-3,4,4',5,6'-pentamethoxychalcone, three 5',6'-(2",2"dimethylpyrano)-polymethoxylated chalcones from N. magnifica var.magnifica (Engler) Emmerich; 4,5 ten polymethoxylated flavonoids, six flavones, three 6,7-(2",2"dimethylpyrano)-flavones and one 6-(3"-hydroxy,3"-methyltrans-but-1"-enyl)-flavone from N. paraensis. 6,7All phytochemical studies on Neoraputia genus have been undertaken in our laboratory and by Arruda et al. 6,7 , and isolation procedures used in these studies should have revealed rutaceous alkaloids, coumarins and limonoids if they had been present.If Emmerich'proposal is correct, that Neoraputia is a Cusparieae member, then this genus can be regarded as a potential source of anthranilate alkaloids.Thus, it would be not surprising if anthranilate alkaloids had remained undiscovered in the Neoraputia because of their low concentrations or due to seasonal variations in the chemical composition of its species.Therefore, it is premature to use the absence of other classes of compounds as an argument to remove Neoraputia to the Citroideae, which produces a considerable number of highly oxygenated flavones. 4,8Clearly much more detailed phytochemical investigations of Neoraputia species will be essential for a better understanding of its chemotaxonomic position in the Rutaceae.In order to establish this we have now undertaken a further investigation of N. paraensis.
Chagas' disease, caused by the protozoan Trypanosoma cruzi, is estimated to affect some 16-18 million people, mostly from South and Central America, where 25% of the total population is at risk (World Health Organisation).Control of the insect vector (Triatoma infestans) in endemic areas has led to the virtual elimination of transmission by insect bites, and, as a consequence, blood transfusion and congenital transmission are currently the major causes for the spread of the disease.Besides low efficacy, the drugs currently available, nifurtimox and benzonidazole, have strong side effects. 9The bloodstream form of the parasite Trypanosoma cruzi has no functional tricarboxylic acid cycle, and it is highly dependent on glycolysis for ATP production. 9This great dependence on glycolysis as a source of energy makes the glycolytic enzymes attractive targets for trypanocidal drug design.Thus, the three dimensional structure of the enzyme was determined. 9Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) catalyses the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Glycosomal GAPDH shows potential target sites with significant differences compared with the homologous human enzyme, and inhibitors have been designed, synthesised, obtained from natural sources, and tested. 9ighly oxygenated flavones from Neoraputia magnifica have shown to be the most actives as glyceraldehyde-3-phosphate dehydrogenase-inhibitor. 5 These data stimulated an investigation of other flavonoids.Thus, in order to find potential lead compounds, flavonoids isolated from Neoraputia paraensis, N. magnifica, Murraya paniculata, Citrus sinensis grafted on C. limon (Rutaceae), Lonchocarpus montanus (Leguminosae) were assayed and evaluated by interaction with the enzyme GAPDH from Trypanosoma cruzi.

Results and Discussion
Our taxonomic interest in the Neoraputia stimulated an investigation of the extracts from stems and leaves of N. paraensis searching for alkaloids, which were detected by the characteristic color with Dragendorff's reagent on TLC plates.Fractions were monitored by 1 H NMR (200 MHz) and ESI-MS/MS and were examined only those which showed features of anthranilate alkaloids and flavonoids absent in the previous investigations.
In a previous paper concerning the chemical systematics of Cusparieae, which Neoraputia has been assigned, 8 the importance of coumarins and anthranilate alkaloids was highlighted.This study brings into focus polyoxygenated flavonoids as another taxonomically useful chemical marker in this group.Many chemists are perhaps interested in alkaloids, coumarins and limonoids from Rutaceae and will rarely identify all of the potentially systematic important classes of compounds presents in the plant, e.g.flavonoids, since they are generally found in most if not all angiosperm plants.Thus, clearly rutaceous flavonoids deserve more attention than they have received so far.
Thus, clearly isoflavonoids and auronoids deserve more attention as glyceraldehyde-3-phosphate dehydrogenaseinhibitors. Highly oxygenated flavones appear to possess the structural requirements for inhibiting trypanosomal GAPDH.However, to develop an effective blocking agent from the natural product lead compounds, it is necessary to determine as precisely as possible, how the tested compounds occupy the active site and at the same time how they make specific interactions with the amino acids of the target enzyme.Therefore, we still have not enough experimental evidence for developing a quantitative understanding of the structural basis of the specificity in the catalytic-site-activity relationships among flavonoids and the enzyme GAPDH.

Experimental
General NMR on a Bruker DRX 400, with TMS as internal standard.HSQC, Heteronuclear Single Quantum Coherence; ESI-MS/MS, low resolution on a triple quadrupole Micromass Quattro LC instrument, equipped with a "Z-spray" ion source; R-HPLC, Recycling High-Performance Liquid Chromatography on a model Shimadzu LC-6AD; detection (Shimadzu SPD-6AV), UV.

Isolation of compounds
Ground stems (2700 g) and leaves (409 g) of Neoraputia paraensis were extracted with n-hexane, then CH 2 Cl 2 and finally with MeOH.Only the concentrated extracts containing alkaloids, which were detected by the characteristic color with Dragendorff's reagent on thinlayer chromatography plates, were worked.Fractions were monitored by 1  The concentrated CH 2 Cl 2 extract was twice flash chromatographed on silica gel, eluting with CH 2 Cl 2 -EtOAc-MeOH gradient and finally with CH 2 Cl 2 -EtOAc gradient affording 4 fractions.Fraction 1 was purified by R-HPLC (the column used was a Shodex Asahipack GS-310P; eluant: MeOH; flow rate: 5.0 mL min -1 ) (detection UV, λ 240 nm) to give 8 (second peak, 2.7 mg), after three cycles of 60 min.Fractions 2, 3 and 4 also were purified by R-HPLC (as above; flow rate: 6.0 mL min -1 ) to give 5 (second peak, 5.0 mg), 6 (second peak, 4.0 mg) and 7 (second peak, 3.0 mg) after three cycles of 60 min, respectively.

Preparation and purification of recombinant T. cruzi GAPDH
TcGAPDH was overexpressed and purified as reported by Souza et al. 9 It is maintained in the Crystallography Laboratory of the University of São Paulo, São Carlos, SP, Brazil.

T. cruzi GAPDH-inhibitory activity
The inhibitory activity was recorded using the reaction medium as above, in a total volume of 1000 µL.
In each case, a blank experiment was performed with 10% DMSO in the reaction medium and was used as the positive control.The specific activity of TcGAPDH was not significantly affected by the presence of 10% DMSO.
Data were means of 3 repetitions and values as percent of control were used as follows: % inhibitory activity = {(U mg -1 control -U mg -1 compound)/U mg -1 control} x 100 Enzymatic inhibition studies have been carried out in the Crystallography Laboratory of the University of São Paulo, São Carlos, SP, Brazil.Some of the potentially active substances, as flavones 6 and 8, can not be assayed with the standard procedure described here, due to problems of solubility in the reaction buffer and absorption of light close to the wavelength used to observe the reaction course (340 nm).To overcome these limitations an alternative technique is being developed, based on calorimetric measurements, similar to procedures adopted with other enzymes. 24 C signals at δ 162.5, 157.3 and 79.1 led to their assignments as C-2, C-4, C-13, respectively.The upfield methine hydrogen signal at δ 5.56 showed correlation with the 13 C signals at δ 105.8, 79.1 and 26.3 permitting the assignment of these signals to C-3, C-13 and C-14/15, respectively.The signals at δ 6.77 and 5.56 were then assigned to H-11 and H-12, respectively.

Table 3 .
Effect of flavonoids a on TcGAPDH activity

Table 4 .
Occurrence of flavonoids evaluated on TcGAPDH activity a Reported for the first time from this species.