Extracts from field peas (Pisum sativum L.) have previously been shown to have a utility to control insect pests. To identify potentially new bioinsecticides in field crops, we describe the fractionation of impure extracts (C8 extracts) derived from protein-rich fractions of commercial pea flour. The activity of separated fractions was determined by a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)], an insect pest of stored products. Bioassay-guided fractionation showed that the triterpenoid saponin fractions were partly responsible for the antifeedant activity of C8 extracts. Soyasaponin I (soyasaponin Bb), isolated from peas and soybeans, and mixtures of soyasaponins, comprised of soyasaponins I-III and isolated from soybeans, were inactive antifeedants, but dehydrosoyasaponin I (the C-22 ketone derivative of soyasaponin I), a minor component found in C8 extracts, was shown to be an active component. Dehydrosoyasaponin I (soyasaponin Be) and soyasaponin VI (soyasaponin betag) coeluted under conditions of silica gel thin-layer chromatography and C18 high-performance liquid chromatography. However, dehydrosoyasaponin I could be isolated from saponin-enriched fractions with a reversed phase column of styrene/divinylbenzene operated at alkaline pH. Phospholipids of the lysolecithin type were also identified in saponin fractions of C8 extracts from peas. Three of the lysolecithins were inactive alone against rice weevils, but mixtures of these phospholipids enhanced the insecticidal activity of dehydrosoyasaponin I.

Crude methanol extracts from four cultivated varieties of mature lentil seeds (Lens culinaris Medik.) were found to possess antifeedant and insecticidal properties in laboratory tests with the rice weevil (Sitophilus oryzae L.), an insect pest of stored products. Flash chromatography with silica gel on active Diaion HP-20 methanol extracts gave flavonol, lysolecithin, soyasaponin, and peptide fractions, as determined by HPLC and electrospray ionization LC/MS. The flavonol fraction was shown by high-resolution NMR experiments to contain a mixture of kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside and, tentatively, kaempferol 3-O-beta-glucopyranosyl(1-->2)-O-[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranoside. These inactive tetraglycosides, although inseparable under the reported HPLC conditions, were detected by NMR spectroscopy in nearly equal proportions. Three lysolecithins were identical to those previously identified in pea extracts. Soyasaponin I (soyasaponin Bb) and soyasaponin VI (soyasaponin betag) were found in Diaion HP-20 methanol extracts. An insecticidal lentil peptide with a mass of 3881 Da, isolated from an Eston variety in small quantities by anion exchange chromatography, was related to the cysteine-rich pea albumin 1b class of botanical insecticides. Binary mixtures of the insecticidal lentil peptide and soybean soyasaponin I were synergistic in tests with S. oryzae.

Methanol soluble insecticidal peptides with masses of 3752, 3757, and 3805 Da, isolated from crude extracts (C8 extracts) derived from the protein-enriched flour of commercial field peas [Pisum sativum (L.)], were purified by reversed phase chromatography and, after reduction and alkylation, were sequenced by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry with the aid of various peptidases. These major peptides were variants of pea albumin 1b (PA1b) with methionine sulfoxide rather than methionine at position 12. Peptide 3752 showed additional variations at positions 29 (valine for isoleucine) and 34 (histidine for asparagine). A minor, 37 amino acid peptide with a molecular mass of 3788 Da was also sequenced and differed from a known PA1b variant at positions 1, 25, and 31. Sequence variants of PA1b with their molecular masses were compiled, and variants that matched the accurate masses of the experimental peptides were used to narrow the search. MALDI postsource decay experiments on pronase fragments helped to confirm the sequences. Whole and dehulled field peas gave insecticidal C8 extracts in the laboratory that were enriched in peptides with masses of 3736, 3741, and 3789 Da, as determined by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry. It was therefore concluded that oxidation of the methionine residues to methionine sulfoxide occurred primarily during the processing of dehulled peas in a mill.

Seed extracts of Desmodium canadense (L.) DC., a native Canadian legume, were examined by HPLC and electrospray ionization mass spectrometry (LC/MS) for the presence of triterpenoid saponins of the oleanene type. An aqueous methanol extract, fractionated by use of a Diaion™ HP-20 macroporous resin, was found to possess soyasaponin III (soyasaponin Bb´) and the 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one conjugate of soyasaponin III (DDMP S-III) as major saponin components. Minor components were identified by LC/MS as C-24 aldehyde (sandosapogenol) derivatives, S-III al and DDMP S-III al. Soyasaponin I (soyasaponin Bb), soyasaponin VI (soyasaponin g) and dehydrosoyasaponin I (soyasaponin Be) were also found as minor components. These components were also detected in extracts obtained by Soxhlet extraction with water followed by partitioning into n-butanol. Extracts from foliage of greenhouse and field origins were essentially free of these saponins. Soyasaponin VI was the main seed saponin of Desmodium illinoense A. Gray whereas soyasaponin III and DDMP S-III were found as minor components. Components corresponding in molecular mass to C-22 glycosides of soyasaponin I and soyasaponin III were also detected in saponin-enriched extracts. Neither species was a good source of alkaloids.