Insecticidal effects of compounds isolated from Trifolium pratense on Myzus persicae, Brevicoryne brassicae and Tetranychus cinnabarinus

Aphids and mites, which cause severe yield reductions, are commonly controlled by commercial insecticides, which has led to serious environmental problems. Research is currently being performed on developing safe and low-toxicity botanical pesticides, which are an effective way to reduce environmental pollution and pesticide residue in food. Trifolium pratense L. is a globally important forage and vulnerable to aphids and mites. The differences in secondary metabolites between infested leaves (ILs) and pest-free leaves (PFLs) of T. pratense were investigated by gas chromatography–mass spectrometry (GC–MS) and high-performance liquid chromatography (HPLC) to determine the chemical defense mechanism. The results revealed significant differences in the contents of fatty acids and isoflavones. The contents of neophytadiene, methyl linoleate, daidzein, genistein, formononetin, and biochanin A in ILs were significantly increased compared with PFLs. The insecticidal compounds were further separated by bioassay-directed fractionation against Myzus persicae Sulzer, Brevicoryne brassicae Linnaeus and Tetranychus cinnabarinus Boisduval. The ethyl acetate (EtOAc) extract had greater insecticidal activity than the petroleum ether (PE) extract. Notably, there was no significant difference between the EtOAc extract and avermectin at 1.00 mg/mL against T. cinnabarinus after 48 h. The following ten compounds were obtained from the PE and EtOAc extracts: prunetin (1), genistein (2), formononetin (3), biochanin A (4), 8-hydroxy-4',7-dimethoxyisoflavane (5), L-maackiain (6), 4-hydroxy-3-methoxy-(6aR,11aR)-8,9-methylenedioxypterocarpan (7), β-sitosterol (8), linoleic acid (9) and linoleic acid monoglyceride (10). Compound 7 exhibited the highest level of insecticidal activity, which was close to that of avermectin. The results indicated that isoflavones were the main active insecticidal components.