Bioactivity-guided isolation of alkamides from a cytotoxic fraction of the ethyl acetate extract of Anacyclus pyrethrum (L.) DC. roots.

Abstract

Introduction. The alcohol extract of Pellitory (Anacyclus pyrethrum) roots has been previously shown to exert anticancer activities on the Human Colorectal Cancer Cell Line (HCT) by targeting apoptosis, metastasis and cell cycle arrest. However, the nature of the cytotoxic molecules associated with this activity remains unexplored. Aims. This study aims to reinvestigate Pellitory root extract as regard to its cytotoxic activity and to proceed to a bioguided fractionation to explore its active fraction and to give new insight in their phytochemical constituents. Methods. Powdered roots were subjected to repeated extraction with Petroleum ether (Pe), Chloroform (Ch), Ethyl acetate (Ea) and Methanol (Me). Pellitory extracts were then screened for cytotoxic activity using the Brine Shrimp Lethality (BSL) bioassay. Results. Ea extract exhibited a marked cytotoxic activity, with LC50 of 249.26 μg/mL in the BSL bioassay. The remaining extracts (Pe,Ch,Me) treated groups exhibited no or low mortality in the range of tested concentrations (1-1000 µg/mL). BSL assay-guided chromatographic fractionation of Ea active Extract revealed a highly cytotoxic fraction (F11) with LC50 of 42.5 µg/mL. Multistep purifications of the active F11 fraction afforded four alkamides, namely N-isobutyldeca-2,4-dienamide or Pellitorine (I), N-propyldodeca- -2,8-dienamide (II), N-isobutyltetradeca-2,4-dienamide (III) and N-propylnona-2,5- -dienamide (IV). Conclusions. This study suggests that cytotoxic activity is localized mainly in the ethyl acetate extract (Ea) of pellitory roots. BSL assay fractionation of this active extract leads to the isolation of four alkamides, including pellitorine (I). While this isobutyl alkamide has previously shown strong cytotoxic activities against human cancer cell lines, the other compounds (II to IV) were not previously reported as cytotoxic. Subsequently, the isolated alkamides will be considered in future study as candidates for in depth in-vitro evaluation of their cytotoxicity against cancer and normal cell lines. Finally, through this study, BSL assay demonstrate again its usefulness as bench-top assay in exploring plant extracts for cytotoxic compounds.

Anacyclus Pyrethrum: A Brief Overview of Phytochemistry, Traditional Uses, and Potential Therapeutic Properties

“…According to a recent study, the ethyl acetate extract used to test the toxicity of the granulated roots showed LC50 = 249.3 gram per milliliters. Four alkamides, including pellitorine, are extracted during the fractionation test of this active extract; this impact may be attributed to their existence as this number is regarded as "toxic" [82].…”

Bioassay With Artemia Salina L.: A Gateway to Understanding the Toxicity of Medicinal Plant Extracts

“…As a result, the need for a practical and reliable method is evident, and this is where the A. salina assay stands out, allowing regular screening of the toxicity of natural compounds. Additionally, it is worth emphasizing that the National Cancer Institute (NCI) has established a significant association between the A. salina assay and the "in vitro" growth inhibition of human tumor cell lines (SILVA et al, 2007;HAMIMED et al, 2018).…”

Phytochemistry, Biological and Pharmacological Activities of the Anacyclus pyrethrum (L.) Lag: A Systematic Review

“…A recent study suggested that the assessment of toxicity is determined mainly in the ethyl acetate extract of the granulated roots, which showed LC 50 = 249.3 μg/mL. This value is considered “toxic”, therefore, the fractionation assay of this active extract leads to the isolation of four alkamides, including pellitorin, and this effect may be associated with their presence [ 100 ].…”