Análises químicas, morfológicas, anatômicas e genéticas comparativas entre três formas de Aristolochia gibertii

Abstract

As part of our ongoing search for bioactive compounds from aristolochiaceous species, we verified that Aristolochia gibertii Hook. exhibit antiplasmodial, antimicrobial, cytotoxic/antitumoral, larvicidal, and insecticidal potentials. This plant is not endemic to Brazil and conspicuous morphological differences have been observed between individuals of this species. Although Aristolochia spp. are a group widely assessed from different points of view, including chemical, phylogenetic, morphologic ones, comparative studies focused below species level are scarce. Since clarifying issues inherent to this gap are relevant within scope of both technological innovation and plant distribution and diversification, the aim of this work was to perform comparative chemical, morphological, anatomical, and genetic analyses between three forms (F1-F3) from A. gibertii, cultivated at the UFGD and originally from three separate geographic localities (F1 Mato Grosso; F2 Selvíria-MS; F3 Dourados-MS). F1 is a typical exemplar of A. gibertii and it was used as reference. Leaves were harvested and immediately dried using silica gel. These samples were subjected to procedures of DNA extraction, and it was followed by amplification and sequencing steps of two chloroplast genes – rcbL and matK. The first rbcL concluded 100% identification between the forms, demonstrating for future doubts that despite different morphology they belong to the same species, while the matK sequence, which according to several authors is a region that can vary, allowed us to confirm F1 and F3 as A gibertii, based on similarity results obtained on the BOLD Systems platform and on the exclusion of classified species that morphologically differ from A. gibertii. In the analysis of the matK region, we observed 17 variable sites between the presents, confirming our hypothesis that in addition to morphological differences in the flowers and genetic variations, as well as in the anatomical part. Through microscopy, it was possible to identify some differences as other epidermal cells of leaves in F3 have wavy anticlinal walls on the adaxial side and sinuous on the abaxial side, while the two have straight walls on both sides. The F1 presented characteristics different from the other forms, presenting characteristics more like druses among others. The F2 had a chemical composition almost identical to that of F1, corroborating the morphological fin-dings which led to the proposal that F1-F3 are the same species. Significant morphological differences were observed between F1-F3, mainly in relation to the size of the flower and length proportion between their upper and lower lips. About 2 g of leaves of each form were extracted with hexanes-EtOAc 1:1 and MeOH, successively, to give 6 extracts, which were analyses by NMR and UHPLC-MS/MS. Diterpenoids and kusunokinin were found as chemical markers of A. gibertii. This dibenzylbutyrolactone lignan was found in all specimens. Curiously, kolavenic acid was not detected in F3, albeit it was the major diterpenoid in F1 and F2. The other chemical similarities and differences between F1 to F3. Thus, our preliminary results indica-ted that F1 to F3 are all A. gibertii, which differ not only morphologically but from a genetic pers-pective and in terms of chemical profile.