Chemical Constituents of Aristolochia giberti

Foram isoladas quatorze substâncias de Aristolochia giberti, entre elas, um novo triterpeno, (-)-onocera-8,8’-diol. Além destas, 3-hidróxipropanoato, acetato e formato foram detectados por técnicas de RMN, o que sugere que estas substâncias sejam derivadas do 2-butinodioato, não detectável por RMN de H. As análises dos óleos essenciais de caules e folhas, por CG-EM e quimiometria, mostraram grande similaridade entre as espécies cultivadas no Brasil e aquelas na Argentina, o que permitiu confirmar a identificação da espécie e diferenciar os óleos de acordo com as partes da planta.


Introduction
Aristolochia species (Aristolochiaceae) are generally sold at Brazilian markets under their popular names, particularly as "One Thousand Man", and little distinction has been made among the species. They have been mainly used in Brazilian traditional medicine as abortifacients, stomachics, antiophidians, antiasthmatics, and expectorants, and recently in slimming therapies. 1,2 Aristolochic acids constitute a class of compounds that are characteristic of the Aristolochia genus. These acids have been associated with Chinese herb nephropathy, which is a kind of severe kidney disease caused by the intake of excessive aristolochic acids. 3 Therefore, it is essential, for health safety and quality control of related Brazilian herbal medicines, to know the chemical composition of these species, among them Aristolochia giberti Hook., and to develop efficient methods for species identification.
The chemical constituents of Brazilian Aristolochiaceae species, such as lignans, have shown antiplasmodial, 4 antimycobacterial, 5 insecticidal, 6,7 anti-inflammatory, and analgesic activities. 8 Despite the significant number of Brazilian Aristolochia species (around 100), the volatile compounds they contain are known for only a few species. 9 In our previous studies on essential oils from roots of 10 Aristolochia species, we investigated the oil composition and correlated them to morphological groups by GC-MS and chemometric analyses, which could also help in the identification of these species. 9 A total of 64 compounds were identified in the analysed essential oils from stems and leaves of A. giberti collected in Argentina and Paraguay, which corresponded to around 77% to 92% of the constituents in the oils. 10,11 Methanolic extracts of A. giberti protected against enzymatic and non-enzymatic lipid peroxidation in microsomal membranes of rat. 12 The goals of the present study were to investigate the chemical composition of the leaf and stem extracts and the nature of the essential oils from stems and leaves of cultivated A. giberti in Brazil, to correlate its oil composition to those reported in the literature for plants collected in Argentina 10 and Paraguay, 11 and then to obtain information about interspecific variability as a function of provenance by using GC-MS and chemometric analyses as tools for plant identification. Vol. 20, No. 9, 2009 Experimental Instrumentation One-dimensional ( 1 H, 13 C, DEPT, and gNOESY) and two-dimensional ( 1 H-1 H gCOSY, gHMQC, gHMBC, and gNOESY) NMR experiments were recorded on a Varian INOVA 500 spectrometer (11.7 T) at 500 MHz ( 1 H) and 126 MHz ( 13 C), using the residual solvents as an internal standard. Mass spectra (electrospray ionization-mass spectroscopy (ESI-MS)) were performed on a Fisons Platform II, and flow injection into the electrospray source was used for ESI-MS. Infrared spectra (IR) were obtained on a Perkin Elmer 1600 FT-IR spectrometer using KBr disks. Ultraviolet (UV) absorptions were measured on a Perkin Elmer UV-Vis Lambda 14P diode array spectrophotometer. Optical rotations were measured on a Perkin Elmer 341-LC polarimeter. Melting points were recorded on a Microquímica MQAPF-301 melting point apparatus and were uncorrected. GC-MS analyses were performed on a Shimadzu GCMS-QP5050A system in EI mode (70 eV) equipped with a split/splitless injector (220 ºC), at a split ratio of 1/10, using a VF-1MS fused-silica capillary column (30 m by 0.25 mm i.d.; film thickness: 0.25 µm). The oven temperature was programmed from 60 ºC (5 min) to 280 ºC at a rate of 4 ºC min -1 and held at this temperature for 10 min. Helium was used as a carrier gas at a flow rate of 0.8 mL min -1 . The injection volume of each sample was 2 µL.

Extraction and isolation of the chemical constituents
The leaves (306.2 g) and stems (1116.5 g) were extracted exhaustively at room temperature with hexane, acetone, and ethanol, successively. The residues were extracted with ethanol in a Soxhlet apparatus and the extracts were individually concentrated.

Essential oils
Stems (30 g) were cut into small pieces and stored at -8 °C until oil extraction. The essential oils were obtained by hydrodistillation in 250 mL H 2 O for 4 h, with simultaneous extraction of the distillate with GC-grade n-hexane (1 mL), which enabled separation of the essential oil in an ice-cooled oil receiver, in a modified Clevenger apparatus to reduce hydrodistillation over-heating artifacts. The oils were collected with the addition of GC-grade n-hexane (1 mL) and dried over anhydrous Na 2 SO 4 . The solutions were then dried over a molecular sieve and analysed by GC-MS. The composition of the volatile constituents was established by GC-MS analyses. Retention indices for all compounds were determined according to the equation proposed by Van den Dool and Kratz, 17 using n-alkanes as standards. Adjusted retention times for each peak were determined by subtracting the retention time of methane from the retention time of each peak. Components were identified based on comparison of their mass spectra with those at the Mass Spectrometry Data Centre, 18 Wiley and NBS Libraries 19 and those described by Adams, 20 as well as by comparison of their retention indices with data in the literature. 9 In several cases, the essential oils were coinjected with compounds that had been previously isolated from Aristolochia species or purchased standard compounds.

Statistical analysis
The agglomerative hierarchical cluster analysis (HCA) and principal component analysis (PCA) were used as statistical methods to suggest the structure of the set and to analyse the variables in relation to the characteristics being studied. To reduce the scattering effects and to compare samples, the chromatograms from obtained oils were normalized by reducing the area under each chromatogram to a value of 1. 21 Overall, 68 characteristics (chemical compounds, of which 67 were identified and one was unknown) were analysed in 10 oils by HCA and PCA (Table  1 and Table S1- Supplementary Information). The chemical compositions were determined from the chromatographic profiles for 2 oils of the Brazilian cultivated species, and taken from data described in the literature for 2 oils from Paraguay, and 6 oils from Argentina. Plots defined by PC1 (score 1) and PC2 (score 2) for the 68 characteristics were obtained for chromatographic data using Pirouette® version 3.11. 22 The results were obtained using an original data matrix X (68 by 10) with 68 variables, 10 samples, 3 optimal factors, 1st derivative. Variances of PC1 (32.6168) and PC2 (3.9711) accounted for 79.90% and 9.73%, respectively, of the total PCA variance.

Results and Discussion
In the oils from cultivated species (fresh material), 17 compounds from the stem and 14 from the leaf were identified (Tables 2 and 3). The data from oils were compared with those described in the literature 10,11 for oils obtained from the stems and leaves of A. giberti, to obtain evidence that could contribute to the identification of species through significant interspecific variability. The variation in the chemical constitutions of the essential oils was examined by taking into account the part of the plant, the year that the plant was collected, and its provenance. Brazilian oils were characterized by the highest concentration of sesquiterpene hydrocarbons in oil from leaves (84.7%) and also the highest concentration of monoterpene hydrocarbons in oil from stems (78.3%). In this study, hierarchical cluster analysis (HCA) was used to search for sample provenance patterns and to create a classification scheme to differentiate plant parts or chemotypes. In this analysis, the data regarding the chemical composition of the oils described in the literature for A. giberti from Argentina (fresh material), 10 involving 36 compounds (one unknown), and Paraguay (dried material), 11 involving 46 compounds, were compared to those obtained in this study (25 compounds) (for further information see Supplementary Data). Based on similarities in the chemical composition, a dendogram was obtained ( Figure 2). According to this dendogram, the oils could be separated into three distinct groups or chemotypes. The first and second groups (I and II) consisted of data of the stem and leaf oils, respectively, obtained from both Brazil and Argentina, and the third group (III) consisted of oils from Paraguay (I, II, and III; similarity index 0.652). The first group was greatly characterized by monoterpenes (a-and β-pinene, sabinene, camphene, tricyclene, and a-thujene). Thus, similar chemical profiles were detected in stem samples collected in different years, which suggests a retention of unique chemical profiles in different populations from Argentina and Brazil.
Similarly, an analysis of the data regarding the chemical composition by PCA (Principal Component Analyses) of the oils was performed to obtain information about the characteristic compounds, which are the most discriminating for the samples observed in the plots (Figures 3 and 4). Except for stem oil from Paraguay (SP), they showed the highest positive scores of  principal component 1 (PC1) and similar PC2 (principal component 2) values for stem oils, which allowed us to differentiate them from the leaf oils ( Figure 3). Oils characterized by the highest positive score signals for PC1 corresponded to stems that showed a predominance of monoterpenes, such as those included in group I by HCA as well as β-myrcene, d-3-carene, and a-phellandrene (Figure 4), whereas the highest negative score signals showed a predominance of oxygenated monoterpenes, such as bornyl acetate and isobornyl formate. Leaf oils from Argentina (LA) and Brazil (LB) were characterized by positive scores of PC2 and by oxygenated sesquiterpenes, such as aristolactone and a-cadinol. Oils from Paraguay (LP and SP) were characterized by negative loading of PC2 for sesquiterpenes, such as d-cadinene and germacrene B (Figure 4). These plots also showed the similarity between stem oils from Argentina and Brazil, which provided evidence regarding the identity of the species and that these oils show insignificant annual chemical variability. However, as shown by HCA, the oils from Paraguay were not well-discriminated by PCA analysis, suggesting that the best results for discriminating the part of the plant and its provenance can be achieved by using fresh material.  (9)], as well as β-sitosterol (10), sequoyitol (11), trigonelline (12), and uracil (13). In addition, 3-hydroxypropanoate, acetate, and formate were detected by NMR techniques, which suggested that they are derived from a natural, yet undetectable compound of 2-butynedioate (14) (Figure 1).
Compounds 1, 2, 4-9, and 11-14 were isolated by partition and chromatographic procedures from the ethanol extracts of the leaves, whereas compounds 2, 3, 6-8, and 10 were obtained by chromatographic procedures from the hexane extract of stems. A. giberti was a rich source of the diterpene (−)-ent-8β-hydroxy-labdan-15-oic acid (2), which represents 8.0% of the ethanol and 21.9% of the ethanol Soxhlet extracts from the leaves.
A colorless amorphous solid (14) was isolated from the ethanol Soxhlet extract of leaves, and its 1 H NMR spectrum did not show any signals for hydrogen in D 2 O, suggesting that, in principle, this solid was an inorganic compound. However, its undetermined melting point (ca. 180 ºC) showed its decomposition as well as its organic character. The 1 H and 13 C NMR spectra in DMSO-d 6 , obtained on consecutive days from the soluble solution resulting from precipitation procedures, showed signals for hydrogens and carbons with diverse intensities, indicating the chemical transformation of compound 14. These spectra together with the results of DEPT, gHMBC, and gHMQC experiments clearly indicated the formation of 3-hydroxypropanoate ( respectively. Both were previously isolated from Aristolochia galeata, 25 and compound 5 was determined to be (-)-eperuic acid. Based on the identity of the observed optical activity of 5 with that reported for its methyl ester derivatives, 26 a 13(S) configuration could be established for 5; while the optical activity of 2 differed from that report for its 13(R) diastereomer methyl ester. 27 Thus, diterpenes 2 and 5 should belong to ent labdane series and has a 13(S) configuration.
The 1 H NMR spectrum of compound 1 was similar to that of 2. The main difference between them was the absence of a doublet corresponding to CH 3 -16 in the spectrum of 1. The 13 C NMR and DEPT (135° and 90°) spectra of 1 showed signals for 15 carbons, including four CH 3 , six CH 2 , two CH, and three quaternary carbons. These data (Table  1), together with the great similarity between 1 H and 13 C NMR spectra of 1 and those of 2 (for further information see Experimental and Supplementary Information, Figures  S1 and S2), as well as the J values determined for the hydrogens by selective proton irradiation, suggested that, except for the substituents at C-9 (side chain), the A and B rings in the structures of both compounds were identical, including their relative configurations.
The HRMS spectra showed quasi-molecular ions [M+Na] + at m/z 469.4018 for 1, which were consistent with the molecular formula C 30 H 54 O 2 + Na. Based on the HRMS and NMR experiments, particularly on the presence of a CH 2 (d C 20.1, d H 0.89), the structure of 1 was determined to be a triterpene, which consisted of two identical sesquiterpene units. Moreover, the correlations observed by gHMBC and 1D gNOESY experiments (irradiating at methyl hydrogen frequencies) allowed us to establish that the monomer units should be linked through C-11, C-11' (Figure 6). The chemical shifts of C-12,12' (d C 30.7) and methyl groups CH 3 20, No. 9, 2009 They determined the metabolic pathways of these two a-alkynoates, in which the triple bonds in both 'invisible substrates' were initially hydrated, and 2-ketobutandioate as well as 3-ketopropanoate were then formed. These authors proposed that both β-keto acids were decarboxylated, resulting in pyruvate and acetaldehyde, respectively. Pyruvate was further hydrolyzed mainly to acetate and formate, whereas minor amounts were reduced to lactate. In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate. 32 Based on this finding, we propose that compound 14 was 2-butynedioate (not detected by 1 H NMR), which, via decarboxylation, could give rise to propynoate, which in turn could give 3-hydroxypropanoate and acetate, and hydration of compound 14 followed by decarboxylation, could lead to acetate and formate ( Figure 8). Thus, the difference between this proposal and those regarding biotransformations is that 14 could give propynoate as one of the intermediates.
Sequoyitol (11), isolated from Aristolochia cymbifera and Aristolochia gigantea, among other species, 1 has been shown to exhibit antidiabetic properties. 33 Sequoyitol, (+)-pinitol, and aristolochic acids are oviposition stimulants for the pipevine swallowtail butterflies, Battus philenor and Atrophaneura alcinous (Papilionidae), which use Aristolochia species as major hosts. 1 Trigonelline (12) is a natural zwitterion isolated from various plants, seeds and the western rock lobster. 23 It has been shown to have hypoglycemic, hypolipidemic, hypocholesterolemic, insulinotropic, and antioxidant activities. 34 These properties are related to the control of the Metabolic Syndrome, a disorder of carbohydrate and lipid metabolism which increases the risk of diabetes and cardiovascular disease. 34 Therefore, trigonelline is a potential natural antidiabetic agent, as well as an antimicrobial and anti-dementia agent. 35,36 Conclusions A. giberti is a rich source of the diterpene and lignans. From this species, 14 compounds were isolated. Among them, a new triterperne, (-)-onocera-8,8'-diol (1), was isolated together with known compounds that are potential agents against several diseases such as diabetes. In addition, 3-hydroxypropanoate, acetate, and formate were also detected and were suggested to be derivatives from 2-butynedioate, which could not be detected by 1 H NMR. Moreover, a total of 25 compounds were identified in the essential oils from stems and leaves. GC-MS and chemometric analyses showed the great similarity between this cultivated species in Brazil and that collected in Argentina, and allowed us to confirm the species identity and to differentiate the oils according to the different parts of the plant.