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An insect antifeedant in catch weed (Galium aparine L.): a strategy for damage avoidance

Masanori Morimoto, Kumiko Tanimoto and Koichiro Komai

Department of Agricultural Chemistry, Kinki University, Nakamachi 3327-204 Nara, Japan. http://nara-kindai.unv.jp/index.html, Email masanori@nara.kindai.ac.jp

Abstract

The insect antifeedant anthraquinone aldehyde nordamnacanthal (1,3-dihydroxyanthraquinone-2-al, ED50 0.12 micro-mol/cm2) was identified in a hexane extract of areal parts of Galium aparine L (Rubiaceae) and calli induced from seedlings of the same species. This anthraquinone had yellowish to orange fluorescence by UV irradiation. Under the UV light, the wounded young plant shows yellowish fluorescence. In the field, some wounded seedling cotyledons had various fluorescences unlike that of the intact plant. The in vivo concentration of nordamnacanthal showed that the tendency to decrease as the plant development. This fact suggested that nordamnacanthal maybe act as a chemical defense system against phytophagous insects during early plant growth stages.

A structure-activity relationship (SAR) determined using a series of alizarin type anthraquinones isolated from Rubiaceae suggested that the aldehyde group on the anthraquinone skeleton was more important than the quinone moiety for the antifeedant activity against phytophagous pest Spodoptera litura F. (Noctuidae). Moreover, because these compounds are natural pigments for dying textiles, we also evaluated their antifeedant activity against a textile pest, carpet beetle (Attagenus japonicus). While nordamnacanthal had strong antifeedant activity against the S. litura, it did not show any antifeedant activity against the carpet beetle. The most effective antifeedant against the carpet beetle was the major constituent in Rubia trictorum, lucidin-3-O-primeveroside, which gave a feeding inhibition rate 88.9% at an applied dose of 1mg/cm2).

Media summary

Anthraquinones from Rubiaceae showed insect antifeedant activity against crop and fabric pest. A large amount of nordamnacanthal was detected in young seedlings as a chemical defense system of Galium aparin.

Key Words

Anthraquinones, Rubiaceae, Galium aparine L., Insect antifeedant, nordamnacanthal, Spodoptera litura, Attagenus japonicus, Chemical defense.

Introduction

The catchweed, Galium aparine L. (Rubiaceae), is an annual weed in Japan. It is not harmed by phytophagous insects in the field and occasionally damages farm crops. Maybe this plant show an allelopathy a general phytophagous insect and crops using by allelochemicals. On the other hand, some members the Rubiaceae have historically been used as a source of natural dyes for textiles. A variety of anthraquinones have been reported in the Rubiaceae (El-Gamal et al. 1995). These constituents showed weak cytotoxic activity (El-Gamal et al. 1997). In this paper, the SAR of anthraquinones as insect antifeedants against the herbivorous insect, Spodoptera litura, and a textile feeding insect, Attagenus japonicus, were determined.

Methods

General

NMR was measured using by TMS as an internal standard. IR spectra were measured using a KBr tablet. TLC was performed on silica gel F254(Merck) using n-hexane-ethyl acetate (3:1). Spots were visualized by fluorescence at 254 and 365 nm or by spraying with 50 % sulfuric acid. Nordamnacanthal has an orange fluorescence by 365 nm irradiation. Alizarin and 2-hydroxymethyl anthraquinone were purchased from Tokyo Kasei Co., Ltd., 2-methylanthraquinone was purchased from Wako Chemical Co., Ltd., and anthraquinone-2-carbonate was purchased from Aldrich Chemical Co., Ltd. The food pigment from Rubia tinctorum was provided by San-Ei Gen F.F.I. Co., Ltd.

Plant materials

Catch weed, G. aparine L., plants and seeds were collected near Kinki University, Nara, Japan, from November to May 1998-2000.

Preparations of test compounds

Fresh aerial parts of G. aparine (3 kg) were extracted with n-hexane for 3 days at 4C, and the resulting extract was concentrated under reduced pressure to give a yellowish green oil (4.19g). The folk drug, the root powder of Rubia akane (2 kg), was also extracted with n-hexane at 4C for 3 days, with the extract concentrated under reduced pressure to give a brown oil (5.37g). The extracts were separated by silica gel column chromatography, eluted with n-hexane-ethyl acetate (3:1), and purified by recrystallization from n-hexane. Lucidin-3-O-primeveroside, purified in crystalline form, was obtained from the food pigment made from the extract of R. tinctorum dissolved in glycerol, keep at 4C for 1 month. Lucidin was prepared by hydrolysis of lucidin-3-O-primeveroside using 6 M HCl aq. at 90C, whereas lucidin-3-O-glucoside was obtained by hydrolysis of lucidin-3-O-primeveroside using 1.2 M HCl aq. Lucidin-3-O-glucoside, dissolved in 1.2 M HCl aq. and hydrolysed at 90C for 1.5h. After neutralization using NaOH aq. we obtained the Lucidin-3-O-glucoside. Lucidin-3-O-primeveroside dissolved in 6 M HCl aq. and hydrolysed at 90C for 4 h obtained lucidin. Anthraquinone-2-al was prepared by oxidation of corresponding anthraquinone alcohol with pyridinium chlorochromate (PCC). The alcohol was dissolved in DCM, and PCC was added to the solution along with 3A molecular sieves keeping for 1 hr. The products were extracted with ethyl acetate. After evaporation of the solvent, the concentrated solution was passed through silica gel with ethyl acetate and recrystallized from hexane give pure crystalline corresponding aldehyde.

Insect Rearing

Common cutworms (Spodoptera litura Lepidoptera, Noctuidae) purchased from Sumika Techno Service Co. Ltd. (Takarazuka, Japan), were reared on an artificial diet (Insecta LF, Nihon Nosan Kogyo Co.) in a controlled room environment at 26.5C and 60 % humidity. Carpet beetles (Attagenus japonicus, Dermestidae) were a gift from Dr. T. Nakashima (Kinki University, Japan) and reared on dried bonito shavings in a controlled room environment at 28C.

Antifeedant bioassay

The experimental setting has been described previously (Morimoto et al. 2002). Leaf-disks, 2 cm in diam., were prepared from fresh sweet potato (Ipomoea batatas) leaves using a cork borer. Two disks were treated with each amount of the test compounds in acetone solution and two other disks that were treated only with acetone as a control. Acetone-insoluble test compounds were applied using an arabic gum paste by the same method. The 4 disks were placed in alternating positions in the same petri dish. After complete removal of the solvent, 15 larvae (3rd instar) were released into the dish. To evaluate antifeedant activity against textile pests, the substrate was changed from sweet potato leaves to swatches of wool fabrics (l cm x 1 cm). The incubation period was 6 hr in the dark for the common cutworm. The slower-feeding carpet beetle larvae were allowed to feed for 2 weeks. AFI (Antifeedant Index) = % of treated disks consumed / (% of treated disks consumed + % of control disks consumed) x 100. A value of less than 30 was considered to reflect antifeedant activity. The ED50 values of the test compounds were calculated by the probit method after changing the AFI value to a feeding inhibition rate.

Results

Insect antifeedant activity was identified in the hexane extract of G. aparine, but the responsible compound was not stable and occurred only in small quantities in the mature plant based on TLC analysis of both the seedling and mature plant extracts. Since a large amount of the same insect antifeedant in the hexane extract of G. aparine was found in the hexane extract of the Rubia akane root, we used R. akane to obtain the insect antifeedant nordamnacanthal. This compound accumulated abundantly in calli induced from seedlings of G. aparine germinated in the dark at 10C. Additionally, the total amount of nordamnacanthal in plants remained essentially constant as plants grew, resulting in a decreased concentration of anthraquinone per plant as measured by HPLC. In a similar manner in the Cyperaceae, it has been reported that the total amount of insect growth inhibitor, JH III changed on each developmental stage (Bede et al. 1999).

Figure 1. Chemical structures of test compounds from Rubiaceae and analogues in this paper.

In the SAR of anthraquinones, nordamnacanthal showed the strongest insect antifeedant activity, with complete inhibition at 83 μg / cm2. Many reports describe quinones and aldehydes have insect antifeedants (Blaney et al., 1987; Morimoto et al., 1999). We have evaluated the importance of an aldehyde group at C-2 of the anthraquinone for antifeedant activity against S. litura. Non-aldehyde anthraquinones and 2-hydroxymethylanthraquinone did not show insect antifeedant activity. The oxidized compound lucidin (1,3-dihydroxy-2-hydroxymethylanthraquinone) did not show any greater antifeedant activity than parent compound. The other test compounds, lucidin-3-O-glucoside and anthraquinone-2-carboxylate, did not show any activity. These results suggested that the aldehyde and hydroxymethyl groups, which supply an active carbon and a hydroxyl group, are important for antifeedant activity against the common cutworms.

Table 1. Antifeedant activities of test anthraquinones against S. litura and A. japonicus

 

S. litura

A. japonicus

Compounds

ED50 (μmol / cm2)

(95% CI)

FIR* (%)

Nordamnacanthal

0.12

(0.111-0.1342)

Inactive

Lucidin

Inactive

 

Inactive

Lucidin-3-O-Glucoside

Inactive

 

Inactive

Lucidin-3-O-Primeveroside

Inactive

 

88.9

Anthraquinone-2-aldehyde

0.75

(0.656-0.825)

Inactive

2-Hydroxymethylanthraquinone

Inactive

 

85.6

Anthraquinone-2-carboxylic acid

Inactive

 

Inactive

2-Methylanthraquinone

3.02

(2.026-4.556)

58.3

Alizarin

Inactive

 

Inactive

*FIR: Feeding Inhibition Rate against carpet beetle larvae, Staining dose: nordamnacanthal and lucidine-derivatives (1 mg / cm2 cloth), other anthraquinones (3 mg / cm2 cloth), Inactive: Antifeedant activity was below 50 % at 0.33 mg/cm2 against S. litura and 3 mg/cm2 against A. japonicus.

Discussion and conclusion

Anthraquinones are natural pigments used in textile dying, and extracts of R. tinctorum protect textiles from the carpet beetle (Nakashima and Doi 1997). However, lucidin-3-O-primeveroside (1 mg/cm2) significantly inhibited feeding, whereas the nordamnacanthal and anthraquinone-2-aldehyde only had weak antifeedant activity against the carpet beetles. This activity disappeared with conversion of the primeveroside to a glucoside. These results suggested that dyes made from R. akane and R. tinctorum may protect against textile pests due to the presence of this primeveroside. Nevertheless these results may be applied to the development of functional textile pigments for the commercial use of anthraquinone dyes. Moreover, these results have revealed some of the chemical ecology of catchweed, and its chemical defense system during plant development.

References

Bede JC, Goodman WG and Tobe, SS (1999). Developmental distribution of insect juvenile hormone III in the sedge, Cyperus iria L. Phytochemistry 52, 1269-1274.

Blaney WM, Simmonds MSJ, Ley SV and Katz, RB (1987). An electrophysiological and behavioural study of insect antifeedant properties of natural and synthetic drimane-related compounds. Physiological Entomology 12, 281-291.

El-Gamal AA, Takeya K, Itokawa H, Halim AF, Amer MM, Saad HEA and Awad SA (1995). Anthraquinones from Galium sinaicum. Phytochemistry 40, 245-251.

El-Gamal AA, Takeya K, Itokawa H, Halim AF, Amer MM and Saad HEA (1997). Lignan bis-glucosides from Galium sinaicum. Phytochemistry 45, 597-600.

Nakashima T and Doi C (1997). Studies on the antimicrobial and moth-proofing efficacies of wool-fabric treated with many kinds vegetable dyes. Journal of Environmental Control Technique 15, 91-105.

Morimoto M, Fujii Y and Komai K (1999). Antifeedants in Cyperaceae: Coumaran and quinones from Cyperus spp. Phytochemistry 51, 605-608.

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