Biologically-guided Isolation of Acaricidal Phytosterols: An in vitro Study against Rhipicephalus (B.) annulatus Ticks Infesting Cattle in Egypt

Aim: To find new and effective natural products to control Rhipicephalus (Boophilus) annulatus infesting cattle in Egypt through biologically-guided study.

Place and Duration of Study: Plant extraction and phytochemical study: Department of Pharmacognosy (2014-2016), Adult and larval immersion tests: Department of Parasitology, Faculty of Veterinary Medicine (2016).

Methodology: Acaricidal activity was evaluated using adult and larval immersion tests of the total alcohol extract of Mesembryanthemum forsskaolii Hochst. Ex. Boiss herb in addition to successive solvent extracts of different polarities (n-hexane, chloroform (CHCl3), and methanol (MeOH). Acaricidal activities were measured by mean number of ticks died and antiparasitic efficacy (%) relative to the negative control. After specifying the most active fraction, chromatographic isolation of the secondary metabolites of the active n-hexane fraction was performed using silica gel columns and SephadexLH-20. Structure elucidation of the isolated compounds was done by extensive 1D/2D NMR study and mass spectroscopy. Re-evaulation of the acaricidal activity of the major isolated compounds was performed to determine the active natural products.

Results: At 10% concentration; n-hexane, CHCl3 and the total alcohol extracts showed 100.00±0.00%, 100.00±0.00% and 86.66±11.15% adulticidal activity respectively compared to 80±10.00% of the acaricide chemical deltamethrin, and 100.00±0.00%, 93.33±2.88% and 96.66±2.88% larvicidal activity respectively compared to 95.00±0.00% of deltamethrin. Only n-hexane fraction retained its 100.00±0.00% mortality when its concentration was reduced to 5%. The polar methanol fraction of M. forsskaolii didn't show any degree of adulticidal or larvicidal activity against the tested tick species. Phytochemical investigation of the n-hexane extract led to the isolation of tricontanol (1), β-amyrin (2), β-sitosterol (3), Sitost-5-ene-3β,7α-diol (4) and β-sitosterol-3-O-glucosid (5). The isolated compounds are being reported for the first time from M. forsskaolii. The major compounds isolated from the most active fraction (n-hexane fraction) were retested for their acaricidal activity. In a concentration of 25 mg/ml; β-sitosterol showed 86.6±5.57% and 91.6±2.88% adulticidal and larvicidal activity respectively while β-sitosterol-3-O-glucosid recorded 76.66±5.57% and 98.33±2.88% respectively.

Conclusion: The n-hexane extract of M. forsskaolii, β-sitosterol and β-sitosterol-3-o-glucoside may be potentially used as natural alternatives in the control of R. annulatus infesting cattle in Egypt. Further studies including field efficacy, persistence and stability need to be done to provide a pharmaceutical preparation for tick control.