Antimicrobial substances from Amazonian Aniba (Lauraceae) species

Abstract Extracts and six isolated substances from Aniba (Lauraceae) Amazonian species A. parviflora, A. panurensis and A. rosaeodora were analysed in vitro to their antibacterial, antiparasitic and antiplasmodial activities. NMR and MS experiments led to the identification of three styrylpyrones (5,6-dihydrokawain [I], 4-methoxy-11,12-methylenedioxy-6-trans-styryl-pyran-2-one [II] and rel-(6R,7S,8S,5′S)-4′-methoxy-8-(11,12-dimethoxyphenyl-7-[6-(4-methoxy-2-pyranyl)]-6-(E)-styryl-1′-oxabicyclo[4,2,0]oct-4′-en-2′-one [III]), a pyridine alkaloid (anibine [IV]) and two kavalactones (tetrahydroyangonin [V] and dihydromethysticin [VI]). The best antibacterial result was observed at the hexane fraction of A. panurensis (MIC 7.8 μg/mL against the three bacteria). Equal MIC were observed by the extract and dichloromethane fraction of A. panurensis against S. simulans and S. aureus; and 15.62 μg/mL against MRSA. Similarly, only A. panurensis extracts showed in vitro activities against Tripanossoma cruzi and Leishmania amazonensis parasites. In Plasmodium falciparum assay, 5,6-dihydrokawain was considered an active antimalarial (14.03 μM), and substances II (132.94 μM) and III (41.84 μM) presented moderate activities.


Introduction
Lauraceae is a basal angiosperm, one of the most endemic in Amazon region, with a complex chemical constitution rich in pyrones, lignans and alkaloids (Custodio and da Veiga 2014), with several pharmacological activities. Aniba is one of its neotropical genus. Previous chemical and pharmacological studies with essential oils from Amazonian Aniba species also showed activities (Alcantara et al. 2010). Recently performed studies have shown a dose dependent and progressive increase skin permeation of antifungal metabolites from Aniba species (Kreutz et al. 2018). At this study, antibacterial, antiparasitic and antiplasmodial in vitro activities of extracts and six substances isolated from Amazonian Aniba parviflora, A. panurensis and A. rosaeodora are present.
Biological activities were already reported to substances I (anti-inflammatory and anti-HIV; Upadhyay et al. 2011;Chou et al. 2013), IV (analeptic activity; Gonçalves et al. 1958) and VI (chemo preventive agent for lung cancer; Puppala et al. 2017). Substances I and II were already described with antimalarial activity (Mccracken et al. 2012) (Figure 1).

Antimicrobial assays
In the preliminary antibacterial screening through the agar diffusion test, only ethanolic extracts and fractions in hexane and dichloromethane of leaves and branches ethanolic extracts of A. panurensis showed inhibition halos, with activity exclusively against Staphylococcus aureus (MIC values at Table S1). Samples showed good activity, and dichloromethane fraction of A. panurensis ethanolic leaves extract exhibited moderate activity (125-250 lg/mL) against S. simulans, S. aureus and MRSA. Branch extracts showed smaller MICs (better activity), when compared with extracts from leaves. The best result was observed at the hexane fraction of A. panurensis, with MIC of 7.8 lg/ mL against the three bacteria, values equivalent to the reference drug TIENAM, and dichloromethane from A. panurensis, with MIC of 7.8 lg/mL against S. simulans and S. aureus; and 15.62 lg/mL against MRSA. Compounds II and III showed no activity. At Plasmodium falciparum (FCR3) antiparasitic assay (Table S2), moderate activities were observed to A. panurensis (branches and leaves) and A. parviflora (leaves) ethanolic extracts, in which the best result was obtained for A. parviflora extract with IC 50 of 29.03 lg/mL. 5,6-dihydrokawain [I] showed the best IC 50 activity of 14.03 lM, but compounds II and III showed only moderate activity.
Anti-T. cruzi assays on Aniba extracts and substances already showed activity (Giongo et al. 2017). At the present study, data obtained (Table S3) evidenced the activity of A. panurensis (leaves and branches) and A. parviflora (leaves) extracts, this one with EC 50 7.2 lg/mL. For the epimastigote form, the most active ethanolic extract was A. panurensis with IC 50 19.6 lg/mL. LLCMK2 cells were treated with extracts to verify cytotoxicity in mammalian cells. The extracts had a selectivity index above one against the relevant clinical form of trypomastigotes, indicating a higher toxicity to the parasites when compared to mammalian cells. L. amazonensis (IC 50 mg/mL) assays on the promastigote form showed that the activity for the three species (A. panurensis, A. roseadora and A. parviflora) were only moderate, 13.2 ± 1.2; 14.6 ± 0.9 and 15.2 ± 1.4 lg/mL, respectively.
The potential observed for the species A. panurensis, A. parviflora and A. roseadora in this work might be associated to the isolated pyrans, as well as the alkaloids or flavonoids, which are classes with described antimicrobial activity. Mass spectrometry experiments MS/MS with extracts of the three species evidenced the presence of alkaloid structures of N-methylcoclaurine, and reticuline. In addition, in A. panurensis and A. parviflora extracts were detected flavonoids: izalpinin and 3,5,7-tri-Omethylgalangine.