Ana Cristina da Silva Pinto

In vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants

In the present study, a quassinoid, neosergeolide, isolated from the roots and stems of Picrolemma sprucei (Simaroubaceae), the indole alkaloids ellipticine and aspidocarpine, isolated from the bark of Aspidosperma vargasii and A. desmanthum (Apocynaceae), respectively, and 4-nerolidylcatechol, isolated from the roots of Pothomorphe peltata (Piperaceae), all presented significant in vitro inhibition (more active than quinine and chloroquine) of the multi-drug resistant K1 strain of Plasmodium falciparum. Neosergeolide presented activity in the nanomolar range. This is the first report on the antimalarial activity of these known, natural compounds. This is also the first report on the isolation of aspidocarpine from A. desmanthum. These compounds are good candidates for pre-clinical tests as novel lead structures with the aim of finding new antimalarial prototypes and lend support to the traditional use of the plants from which these compounds are derived.

Screening of plants found in Amazonas State for lethality towards brine shrimp

226 methanol and water extracts representing 74 mainly native plant species found in Amazonas State, Brazil, were tested at a standard concentration of 500 μg/mL for lethality towards larvae of the brine shrimp species Artemia franciscana. Several cytotoxic plant species were identified in this work: Aspidosperma marcgravianum, A. nitidum, Croton cajucara, Citrus limetta, Geissospermum argenteum, Minquartia guianensis, Piper aduncum, P. amapense, P. capitarianum, P. tuberculatum and Protium aracouchini. The results were analyzed within the context of the available traditional knowledge and uses for these plants.

Screening of plants found in the State of Amazonas, Brazil for activity against Aedes aegypti larvae

Ethanol, methanol and water extracts representing mostly native plant species found in the Amazon region were prepared, respectively, by maceration, continuous liquid-solid extraction and infusion, followed by evaporation and freeze-drying. The freeze-dried extracts were tested for lethality toward Aedes aegypti larvae at test concentrations of 500 mg / mL. In general, methanol extracts exhibited the greatest larvicidal activity. The following 7 methanol extracts of (the parts of) the indicated plant species were the most active, resulting in 100% mortality in A. aegypti larvae: Tapura amazonica Poepp. (root), Piper aduncum L. (leaf and root), P. tuberculatum Jacq. (leaf, fruit and branch). and Simaba polyphylla (Cavalcante) W.W. Thomas (branch).

Chemical Composition and Larvicidal Activity against Aedes aegypti Larvae of Essential Oils from Four Guarea Species

The essential oils of four Guarea species collected at Manaus (Amazonas, Brazil) were obtained by hydrodistillation and analyzed by GC-MS. Except for one diterpene detected, the compounds identified in the essential oils were hydrocarbons and oxygenated sesquiterpenes. The major sesquiterpenes were α-santalene (26.26%) and α-copaene (14.61%) from G. convergens branches; caryophyllene epoxide (40.91%) and humulene epoxide II (14.43%) from G. humaitensis branches; cis-caryophyllene (33.37%) and α-trans-bergamotene (11.88%) from G. scabra leaves; caryophyllene epoxide (36.54%) in leaves and spathulenol (14.34%) in branches from G. silvatica. The diterpene kaurene (15.61%) was found in G. silvatica leaves. Larvicidal activity assay of essential oils against third-instar Aedes aegypti larvae revealed that at higher concentrations (500 and 250 μg/mL), all the essential oils caused 100% mortality after 24 h of exposure. The most active essential oils were those of G. humaitensis branches (LC50 48.6 μg/mL), G. scabra leaves (LC50 98.6 μg/mL) and G. silvatica (LC50 117.9 μg/mL). The differences in the toxicity of essential oils of Guarea species on A. aegypti are due to qualitative and quantitative variations of the components, therefore the larvicidal effect may be due to higher amount of the sesquiterpenes with caryophyllane skeleton.

New antimalarial and cytotoxic 4-nerolidylcatechol derivatives.

4-Nerolidylcatechol (1) was isolated from cultivated Pothomorphe peltata root on a multigram scale using straight-forward solvent extraction-column chromatography. New semi-synthetic derivatives of 1 were prepared and tested in vitro against multidrug-resistant Plasmodium falciparum K1 strain. Mono-O-methyl, mono-O-benzyl, O,O-dibenzyl and O,O-dibenzoyl derivatives 2-8 exhibited IC(50) in the 0.67-22.52 microM range. Mono-O-methyl ethers 6 and 7 inhibited the in vitro growth of human tumor cell lines HCT-8 (colon carcinoma), SF-295 (central nervous system), LH-60 (human myeloblastic leukemia) and MDA/MB-435 (melanoma). In general, derivatives 2-8 are more stable to light, air and pH at ambient temperatures than their labile, natural precursor 1. These derivatives provide leads for the development of a novel class of antimalarial drugs with enhanced chemical and pharmacological properties.

In Vivo and In Vitro Antimalarial Activity of 4‐Nerolidylcatechol

4‐Nerolidylcatechol (4‐NC) isolated from Piper peltatum L. (Piperaceae) was evaluated for in vitro antiplasmodial activity against Plasmodium falciparum (cultures of both standard CQR (K1) and CQS (3D7) strains and two Amazonian field isolates) and for in vivo antimalarial activity using the Plasmodium berghei‐murine model. 4‐NC exhibits significant in vitro and moderate in vivo antiplasmodial activity. 4‐NC administered orally and subcutaneously at doses of 200, 400 and 600 mg/kg/day suppressed the growth of P. berghei by up to 63% after four daily treatments (days 1–4). Also, 4‐NC exhibited important in vitro antiplasmodial activity against both standard and field P. falciparum strains in which 50% inhibition of parasite growth (IC50) was produced at concentrations of 0.05–2.11 μg/mL and depended upon the parasite strain. Interestingly, healthy (non‐infected) mice that received 4‐NC orally presented (denatured) blood plasma which exhibited significant in vitro activity against P. falciparum. This is evidence that mouse metabolism allows 4‐NC or active metabolites to enter the blood. Further chemical and pharmacological studies are necessary to confirm the potential of 4‐NC as a new antimalarial prototype. Copyright

Median Lethal Concentrations of Amazonian Plant Extracts in the Brine Shrimp Assay

In the current study, median lethal concentrations (LC50) were obtained in the brine shrimp assay (BSA) for 31 methanol and water plant extracts representing 21 Amazonian plant species, most of which are used in traditional medicine. The following species presented extracts with LC50 values less than 50 µg/ml: Croton cajucara Benth., Micrandropsis scleroxylon W.A. Rodrigues, Piper aduncum L., P. hostmannianum (Miq.) C. DC., P. tuberculatum Jacq., Protium aracouchini (Aubl.) Marchand, and Tapura amazonica Poepp. The methanol extract of T. amazonica Poepp., a species known to be toxic according to ethnobotanic reports, was the most active (LC50 1.2 µg/ml). The data presented are considered useful as general toxicity parameters, and their relevance in the context of discovery of new antitumor agents and potential sources of (new) bioactive compounds in the Amazonian flora is discussed.

Stability and Antioxidant Activity of Semi-synthetic Derivatives of 4-Nerolidylcatechol

4-nerolidylcatechol (4-NC) is an unstable natural product that exhibits important antioxidant, anti-inflammatory and other properties. It is readily obtainable on a multi-gram scale through straightforward solvent extraction of the roots of cultivated Piper peltatum or P. umbellatum, followed by column chromatography on the resulting extract. Semi-synthetic derivatives of 4-NC with one or two substituent groups (methyl, acetyl, benzyl, benzoyl) on the O atoms have been introduced that have increased stability compared to 4-NC and significant in vitro inhibitory activity against the human malaria parasite Plasmodium falciparum. Antioxidant and anti-inflammatory properties may be important for the antiplasmodial mode of action of 4-NC derivatives. Thus, we decided to investigate the antioxidant properties, cytotoxicity and stability of 4-NC derivatives as a means to explore the potential utility of these compounds. 4-NC showed high antioxidant activity in the DPPH and ABTS assays and in 3T3-L1 cells (mouse embryonic fibroblast), however 4-NC was more cytotoxic (IC50 = 31.4 µM) and more unstable than its derivatives and lost more than 80% of its antioxidant activity upon storage in solution at −20 °C for 30 days. DMSO solutions of mono-O-substituted derivatives of 4-NC exhibited antioxidant activity and radical scavenging activity in the DPPH and ABTS assays that was comparable to that of BHA and BHT. In the cell-based antioxidant model, most DMSO solutions of derivatives of 4-NC were less active on day 1 than 4-NC, quercetin and BHA and more active antioxidants than BHT. After storage for 30 days at −20 °C, DMSO solutions of most of the derivatives of 4-NC were more stable and exhibited more antioxidant activity than 4-NC, quercetin and BHA and exhibited comparable antioxidant activity to BHT. These findings point to the potential of derivatives of 4-NC as antioxidant compounds.

Piper peltatum: Biomass and 4-Nerolidylcatechol Production

Piper peltatum L. is used for the treatment of inflammation, malaria, and other ailments. 4-Nerolidylcatechol (4-NC) is a valuable natural product that has important anti-inflammatory, antimalarial, and antioxidant properties. 4-NC is a component of P. peltatum and P. umbellatum extracts, which are used in cosmetics. The aim of this work was to evaluate the production of plant biomass and the production of 4-NC in roots of cultivated P. peltatum over a full life cycle. Seedlings were produced in a greenhouse and then transplanted. The weight of dry plant parts (leaves, stems, roots, and inflorescences); numbers of stems, leaves, and inflorescences; and the leaf-to-stem ratio were evaluated at intervals of 60 days after transplanting (DAT). Extracts were prepared using 1:1 ethanol-chloroform and an ultrasound bath. Roots, leaves, and inflorescences contained 4-NC according to TLC photodensitometry analysis. Quantification of 4-NC in root extracts was performed using HPLC-DAD analysis. Per-hectare production of 4-NC by roots was estimated based on quantitative HPLC analysis and biomass data. Optimal per-hectare yields of 4-NC were obtained by harvesting roots between 350 and 400 DAT. In this period, the average yield was 27 kg 4-NC per hectare. Importantly, at the time of maximal overall production of root biomass (470 DAT), there was a decrease in the production of 4-NC (23.8 kg/ha), probably due to the onset of senescence.

Larvicidal Activities against Aedes aegypti and Culex quinquefasciatus of Some Extracts from Amazon Edible Fruits

The Amazon Forest encompasses a vast diversity of edible fruits, which are very appreciated by the local population. There are few studies describing the chemical composition or the biological activities for most of the Amazonian edible fruits. On the other hand, the Brazilian Amazon region is also endemic area for most of the tropical diseases in the country. One of the most effective control is the vector control at larval stage, especially with botanicals. The larvicidal activities against Aedes aegypti and Culex quinquefasciatus have been determined for the extracts and fractions of five Amazonian edible fruits: pataua ( Oenocarpus bataua ), bacaba ( Oenocarpus bacaba ), uxi ( Endopleura uchi ), tucuma ( Astrocaryum aculeatum ) and piassava ( Leopoldinia piassaba ). Lethality assay of extracts and fractions against third-instar Aedes aegypti and Culex quinquefasciatus larvae revealed that at higher concentrations (500 μg.mL -1 ), all the extracts caused 100% mortality after 48 h of exposure to larvae Culex quinquefasciatus . The tested extracts and fractions exhibited larvicidal effects against Culex quinquefasciatus with LC 50 values between 29.7 and 157.2 μg.mL -1 . The results obtained should be of value in search for new natural larvicidal compounds.