HPLC-quantification of yangambin and cytotoxicity of Ocotea duckei extract against fibroblast cell line L929 for studies in neglected diseases such as Leishmaniasis

Leishmaniasis is a neglected poverty-related disease and, currently, is considered to be endemic in 88 countries. The high price and the low access to medicines have led the research for other substances to treat this disease. The important activity of several lignans, especially yangambin, has been observed recently and the Ocotea duckei plant extract, from Brazilian biodiversity, has shown promising activity against L. amazonensis and L. chagasi. Therefore, the objective of this study was to quantify yangambin by HPLC and to obtain a screening of the cytotoxicity of this extract in a fibroblast cell line for further in vivo studies. The quantification of yangambin in the plant extract was performed using HPLC (C18 column and MeCN:H₂O (50:50) as mobile phase), using a yangambin standard. Mouse fibroblasts (L929) at 5 × 10⁴/mL were inoculated in 96 well microplate. After 24 hours, they were added with a solution containing the extract (6.25 – 200 µg/mL). At specified periods (30 minutes, 8h and 24 hours) the culture medium was discarded and the coloration was performed with neutral red. The HPLC assay demonstrated that the extract contained 314,6 mg yangambin per gram dry weight. Regarding toxicity, the 6.25 – 200 µg/mL concentration range studied demonstrated not to be cytotoxic to the fibroblast cell line. Researches have pointed that the IC₅₀ for L. amazonensis and L. chagasi is 143,74 µg/mL and 135,66 µg/mL, respectively, and the inhibition were higher than Glucantime® [1]. Other studies [2] demonstrated that concentrations of pure yangambin (50 and 65 µg/mL), changed the morphological and physiological promastigotes forms. Knowing this and based on the fact that the higher concentration tested was not cytotoxic (200 µg/mL) and it contains 62,92 µg/mL of yangambin, the Ocotea duckei extract may be an interesting object for further studies.

References:

[1] Monte-Neto et al.; Z. Naturforsch. 62c, 348 – 352 (2007)

[2] Monte Neto et al; Experimental Parasitology 127, 215 – 221 (2011)