Isolation and Identification of an Insecticidal Soyasaponin from Field

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Chapter 15

Isolation and Identification of an Insecticidal Soyasaponin from Field Pea Extracts 1

Wesley G . Taylor and Paul G . Fields

2

1

Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan S7N 0X2, Canada Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba R3T 2M9, Canada 2

Dehydrosoyasaponin I was identified as a minor component with antifeedant and insecticidal properties in extracts from yellow field peas (Pisum sativum L.). Chromatographic fractionation of crude methanolic extracts (C8 extracts) from commercial protein-rich pea flour yielded fractions containing soyasaponins and phospholipids in addition to other fractions containing the insecticidal pea albumin 1b family of cysteinerich plant peptides. Activity of the saponin fractions was determined by a flour disk bioassay with the rice weevil [Sitophilus oryzae (L.)], an insect pest of stored products. Synergists of dehydrosoyasaponin I were also present in C8 extracts, in the form of three phospholipids of the lysolecithin (lyso-phosphatidylcholine) type.

© 2006 American Chemical Society Rimando and Duke; Natural Products for Pest Management ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


195 Certain legume plants, including seeds of the pea (Pisum sativum L.), are toxic to insects (1-4). Bodnaryk et al. (5) showed that commercial flour from field peas was insecticidal to stored-product insects. Protein-rich pea flour was more effective against stored-product insects than starch- or fibre-rich fractions (5, 6). A n extraction procedure was developed with hot 80% methanol (20% water) to obtain crude insecticidal pea extracts. The aqueous methanol extracts from defatted, protein-rich flour were partially purified with reversed phase C8 silica (5). Activity was found in fractions obtained by elution of the C8 silica column with 100% methanol. These C8 extracts displayed antifeedant and insecticidal activity against rice weevil [Sitophilus oryzae (L.)] and other stored-product insects but the active ingredients of C8 extracts were not identified. In granary trials (7), protein-rich pea flour at a concentration of 0.1% reduced S. oryzae by 90% and rusty grain beetle [Cryptolestes ferrugineus (Stephens)] and red flour beetle [Tribolium castaneum (Herbst)] by 70%. However, this concentration was probably too high for practical use as a grain protectant. Insect pests infesting stored grains are primarily controlled by organophosphorus insecticides, ozone-depleting methyl bromide and toxic phosphine gas (8). Alternate approaches for insect control with safe and effective natural products are needed, especially from food-grade materials. The purpose of our research was to isolate and identify the anti-insect components of pea extracts derived from air-classified protein-rich field pea flour. This chapter describes bioassay-guided fractionation by silica gel chromatography of C8 extracts, characterizing active components contained in fractions of intermediate polarity (9). Highly polar end fractions from C8 extracts were found (10, 11) to contain mixtures of cysteine-rich peptides of the pea albumin l b ( P A l b ) type (72). Some of these P A l b peptides had previously been extracted from peas with acetate buffer and were shown to be effective against stored-product insect pests (13).

Experimental C8 Extracts and Insect Bioassays Protein-rich pea flour, obtained by an air-classification process (14), was supplied by Parrheim Foods Limited (Saskatoon, Canada). The flour was extracted in the laboratory with 80% methanol (5) (Figure 1). Utilizing two C8 SepPak Vac™ cartridges per 100 g of flour, the C8 extracts in methanol were combined and concentrated to dryness with a centrifugal Savant evaporator. Starting with 100 grams of defatted protein-rich flour, a beige C8 powder was obtained in 0.7-0.9% yield. Antifeedant activity was assessed with a flour disk bioassay (75) with 70% ethanol as solvent. Twenty five adult S. oryzae (1-2 weeks old) were held on five wheat flour disks for three days at 30 °C, 70% relative humidity. Flour disks (ca. 0.1 g/disk) were weighed before and after exposure to the insects. Antifeedant activity was determined by expressing consumption of treated disks

Rimando and Duke; Natural Products for Pest Management ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

196 field peas

starch, fibre

air classification process


protein-rich flour

chloroformsoluble fraction

chloroform defatting

defatted proteinrich fraction

80%MeOHinsoluble cake

aqueous methanol extraction (70° C, 5 minutes)

methanol filtrate (> 70% water)

50%MeOH wash

C8 silica Sep-Pak Vac™ cartridges

C8 extract (in MeOH) solvent evaporation MeOH

C8 powder Figure 1.

Processing steps employed during isolation of insecticidal C8 extracts.


197


as a percentage of control disks (70% ethanol). Positive controls using the same C8 extract were run with each bioassay. Insecticidal activity was assessed as median survival time by Kaplan-Meier survival analysis (log-rank) using SigmaStat (SPSS Inc., Chicago, IL). After weighing the disks, S. oryzae and flour disks were returned to the Petri dishes and survivors noted each day until the insects had been on the disks for a total of 14 days. A close correlation was previously found between antifeedant activity and toxicity of pea extracts (5, 16).

Fractionation of C 8 Powder Initially, samples of C8 extracts were separated by column chromatography with silica gel and chloroform-methanol mixtures. The C8 powder gave two chromatographically distinct, medium to high polarity bands that showed good antifeedant activity. The first active band, appearing in the 40% chloroform (60% methanol) fraction and designated C8-la, represented about 10% of the mass of applied C8. It reduced food consumption to 15% of untreated samples. The second active, highly polar band eluted gradually with 80-100% methanol and was collected as five separate fractions at the end of the experiment. These end fractions, collectively representing about 25% of the applied C8, gave food consumption values of 24-45%. The most active of these five fractions, designated as C8-lb, was compared in a dose-response experiment to C 8 - l a and to impure C8 (Figure 2). Samples of C8, C 8 - l a and C8-lb, each with practically identical antifeedant activity, were spotted on analytical T L C plates ( E M Science plastic sheets, 0.2 mm layer thickness) and developed with a mixture designated as solvent system 1 (the lower layer of chloroform-methanol-water: 65-35-10, by volume). B y employing ultraviolet light plus T L C detection reagents (applied as sprays to the developed plates and exemplified by ninhydrin and Liebermann-Burchard reagents), it could be demonstrated that C8, C 8 - l a and C 8 - l b were complex mixtures. However, certain of the T L C spots in the sample of C 8 - l a gave a grey colored response to Liebermann-Burchard, indicative of the presence of triterpene saponins such as soyasaponin I (17) whereas most of the spots from C 8 - l b were positive to ninhydrin, indicating that this fraction probably contained peptides (18). This implied that at least two chemically distinct insecticidal components were present in the C8 extract. A technique was sought that was faster than column chromatography to fractionate the C8 mixture into the equivalent of C 8 - l a and C8-lb, ensuring that there was a clear separation between these insect-active fractions. This objective was achieved by flash chromatography using a F L A S H 40 M ™ apparatus (Biotage Inc., Charlottesville, V A ) equipped with a prepacked 90 gram ( 4 x 1 5



198

100-,

Concentration (mg/200 mg flour) (log scale) Figure 2. Comparison of dose-response curves in antifeedant bioassays with S. oryzae using a crude C8 extract and partially purified C8-la and C8-lb extracts. (Reproducedfrom reference 9. Copyright 2004 American Chemical Society.)


199 cm) KP-Sil™ (Biotage) cartridge (32-63 :m, 60 Δ silica). Flow rate was maintained at 20 ml/min with solvent system 1. The properties of the collected fractions are shown (Table I).


Table I. Fractionation of a C8 Extract (250 mg) by Flash Chromatography with a Silica Cartridge. Fraction 1 2 (C8-2a) 3 4(C8-2b) c

a

RF"

>0.5 0.25-0.5 14 d > 14 d 6.7 ± 0.3 b 9.0 ± 0.5 c > 14 d > 14 d > 14 d > 14 d

Food consumption, expressed as % of control, in the S. oryzae bioassay. Samples were tested at the indicated amounts in 200 mg of flour.

a

Kaplan-Meier survival analysis was used to estimate the median survival times and multiple comparisons were made with the Holm-Sidak method, Ρ = 0.05. Medians followed by a different letter are significantly different. All insects in controls survived to 14 days. c

Purified by methods a, c, b and d of reference 9.

d

Isolated by RPC with an AKTAExplorer 100 LC (Amersham Biosciences).

Source: Reproducedfromreference 9. Copyright 2004 American Chemical Society.

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Isolation and Identification of an Insecticidal Soyasaponin from Field

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