Optimization of extraction method and evaluation of antileishmanial activity of oil and nanoemulsions of Pterodon pubescens benth. fruit extracts.

Currently, leishmaniasis is difficult to manage owing to the limited choice and high toxicity of available drugs, and emergence of drug-resistant protozoa. Medicinal plants, which produce various bioactive molecules, can help counter this global shortage. In this study, we prepared Pterodon pubescens fruit extracts, which show antileishmanial activity, and developed a nanoemulsion of the optimized extract to improve its performance. The extracts were prepared using conventional methods and a supercritical fluid method and were tested for activity against Leishmania amazonensis promastigotes and amastigotes. The two most effective extracts were formulated as nanoemulsions. Although both extracts showed cytotoxicity, the supercritical extracts were more effective against L. amazonensis promastigotes and amastigotes than conventional extracts were. This was attributed to the high content of the geranylgeraniol derivative in the supercritical extracts. The nanoemulsions showed a better selectivity index and significantly improved activity against parasites (IC50: 2.7 µg/mL for nanoemulsion of hexane extract; IC50: 1.9 µg/mL for nanoemulsion of supercritical extract) compared to the Miltefosine standard (0.7 µg/mL). This could be due to the smaller droplets of the supercritical extracts, allowing better penetration. In conclusion, the extracts showed promise in in vitro tests, and could be used as a leishmaniasis treatment.

Determination by GC-MS-SIM of furanoditerpenes in Pterodon pubescens Benth.: development and validation.

The crude hydroalcoholic extract from fruits of P. pubescens is widely used because of its anti-rheumatic, antinociceptive and anti-inflammatory activities. Furanoditerpenes have a vouacapan skeleton and are involved with the pharmacological activity of the oil extracted from P. pubescens fruits. Furanoditerpenes methyl 6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate and methyl 6α-hydroxy-7ß-acetoxyvouacapan-17ß-oate from P. pubescens were isolated and identified. The present study developed and validated a GC-MS-SIM method for the separation and quantification of vouacapan constituents in a semipurified extract from P. pubescens fruits. The GC-MS analyses were carried out using a system equipped with a HP-5 capillary column (30 m × 0.25 mm). Temperature program: 100°C (4°C min(-1))-270°C (5 min), injector 260°C, detector 270°C. Helium was used as the carrier gas (0.7 bar, 1 mL min(-1)). The MS was taken at 70 eV. Scanning speed was 0.5 scans s(-1), from 50 to 650. Sample volume was 1 µL. Split 1:20. Analyses for validation of methodology were conducted by GC-MS-SIM (Single Ion Monitoring), where the ions monitored were 131, 145 and 146 (between 43 and 44.5 min), 105, 145 and 197 (from 44.5 to 45.3 min) and 131, 178 and 312 (from 45.3 to 48.5 min).Validation of the analytical method was based on the following parameters: linearity, robustness, limits of detection and quantification, precision (within-day and between-day variabilities), recovery and stability. The method was linear over a range of 12.81-2.56 µÎ³µΛ(-1) of vouacapan 1, 112.78-22.56 µg mL(-1) of vouacapan 2, and 333.34-66.67 µg mL(-1) of vouacapans 3 and 4, with detection limits of 0.39, 3.45 and 9,44 µg mL(-1) and quantification limits of 1.19, 10.47 and 28.62 µg mL(-1), respectively. Recovery values were 100.69%, 97.48% and 96.98% for vouacapans 1, 2 and 3-4, respectively. Thus, the method was efficient to separate and quantify furanoditerpenes in the extract or fraction.