JPH0393800A - Growth inhibitor for larva of azuki bean weevil or the like extracted from been seed - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to the production of glycoproteins such as Adzuki bean weevil and Japanese bean weevil, which are extracted from kidney bean seeds and have a molecular weight of about 48,000 and an isoelectric point of pH 4.46. The present invention relates to a larval growth inhibitor, a method for producing the inhibitor, and the larva growth inhibitor containing the inhibitor as an active ingredient.

[Prior art] Callosobruchus chinensi of the family Bruchidae
s Linn! ), Callo
sobruchus maculatus Pabri
The larvae of bean seeds such as ciuS) cause feeding damage to many bean seeds, but the bean beetles and beetles in particular cause great damage to the azuki beans during storage. Therefore, research is continuing to find inhibitors that are highly safe for humans and can effectively suppress these pests.

Adzuki bean weevil larvae grow in 16 different species of bean seeds under experimental conditions, but in common bean (Phaseolus
vulgaris L. ) does not grow in seeds. The same thing occurs with the bean weevil larvae, due to the presence of substances that are toxic to these insects but not to humans (Reference 1: References cited herein do not cite the invention. (They are summarized at the end of the detailed description section). These larvae die in the first instar in the bean seeds.

Janzen et al. (Reference 2) reported that black beans (Ph
It is stated that the growth inhibition of the Japanese bean weevil caused by Aseoluus vulgaris is due to the presence of phytohemagglutinin (lectin) and not to the tribucin inhibitor. On the other hand, Gatehou
SE et al. (Reference 3) reported that various cowpeas (Vigna ung
uiculata L. ) concluded that the resistance was due to increased amounts of trypsin inhibitors.

Tanaka et al. (Reference 4) reported that crude lectin obtained from kidney bean showed growth inhibitory activity against kidney bean (resistant) and beans (resistant).
It was found that there is a certain relationship between the unit activity and content of crude lectin in susceptibility) and growth inhibitory activity, whereas lectin purified by affinity chromatography had no activity.

α from seeds of Phaseolus vulgaris
- Amylase inhibitors are known to inhibit α-amylase in some cereal insects in vitro (P
owers &Culbertson; Reference 5).

On the other hand, Gatehouse et al. (Reference 6) correlated the inhibition of α-amylase derived from the Japanese bean weevil with an α-amylase inhibitor obtained from wheat and the inhibition of its larval growth.

Furthermore, fshimoto and KiLamura (Reference 7) found that a lectin preparation from kidney beans had no effect on adzuki bean weevil larvae, and isolated an α-amylase inhibitor that was highly toxic to the larvae. They note that this inhibitor is a glycoprotein with a molecular weight of approximately 1oo,ooo, but provide no further experimental evidence.

[Problem to be Solved by the Invention] The present inventor has been working independently on the above problem for several years. As a result, a growth-inhibiting substance for larvae such as adzuki bean weevils, which is a glycoprotein with α-amylase inhibitory activity and an isoelectric point pH of 4.46, was isolated from kidney bean seeds. That is, the present invention provides a growth inhibitory substance, a method for producing the inhibitory substance, and a growth inhibitor containing the inhibitory substance as an effective component.

[Means for Solving the Problems] The growth inhibitory substance of the present invention can be obtained by subjecting a water-soluble extract of common bean seeds to gel filtration chromatography while assaying the growth inhibitory activity of adzuki bean weevil larvae, as described in Examples below. It was isolated by fractionation. That is, the growth inhibitory substance of the present invention can be obtained by (a) extracting kidney bean seed powder with a phosphate buffer, (b) fractionating the extract by gel filtration chromatography using cross-linked agarose as a carrier, and (c) fractionating the extract. It can be produced by further fractionating one of the fractions by gel filtration chromatography using cross-linked dextran as a carrier.

Extraction of kidney bean seed powder is carried out by crushing kidney bean seeds and extracting them with a phosphate buffer at room temperature or under heating. Next, centrifuge and collect the supernatant.
This crude extract was transferred to a cross-linked agarose, e.g. Sepharo
Apply to a gel filtration ram such as se 6B and elute with acetate buffer. Elute at a constant rate, monitor protein content and divide into 4 fractions. This third fraction was applied to a gel filtration column of cross-linked dextran, e.g. Sephadex G-75, eluted with acetate buffer and filtered at 280 n
A, B, C, corresponding to the four absorption peaks at a+
Fractionate into 4 fractions (D). This fraction B was dialyzed against distilled water,
The growth inhibitory substance of the present invention can be obtained by freeze-drying.

Furthermore, the growth inhibitor of the present invention is produced by making the above-mentioned growth inhibiting substance into an effective amount and mixing it with various carriers to form a dosage form suitable for various application methods. The carriers used may be those known in the art and the application of growth inhibitors may be conventional.

[Effects of the Invention] The growth-inhibiting substance of the present invention is extracted from kidney beans, and is toxic to larvae such as adzuki bean weevil, but non-toxic to humans. Therefore, stored azuki beans and the like can be safely protected from pests.

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.

[Example Raw Materials and Method 1, Bioassay Method As shown in the process chart 1 below, the bioassay was performed by modifying the method of Lshii (Reference 1). Each component of kidney bean was mixed with adzuki bean flour and water, and this mixture was made into artificial beans. Adzuki bean weevils were allowed to lay eggs on the artificial beans, and 10 to 12 days later, the beans were crushed and the surviving larvae and their instars were examined using a microscope.

(Hereinafter, blank space) Process diagram 1 Preparation of artificial beans for bioassay 10 artificial beans (50 sins each) with adzuki bean powder mixed powder 0.59 and kidney beans (50 sins each) Bioassay 2. Extraction of kidney beans The extraction of kidney beans was carried out as shown in process diagram 2 below.

2.1. Preparation of Crude Extract Finely ground kidney beans (80 mesh: L; 40 g) were prepared in a 30+ nM phosphate buffer [pH 7. 5; 0. 4M NaC1!
(1 hour at 30'C). Then 7
The extract was separated from the slurry by centrifugation at 5.000xg for 15 minutes, and the supernatant was collected at 25. Centrifugation was performed for 30 minutes at 0 0 0 X9 (twice). The total amount of precipitate (residue) is 2
It was 4.139. This supernatant (referred to as crude extract) was
Dialyzed against distilled water at °C and lyophilized to 7.039 (
An extract of 40 y-eq) was obtained for bean.

2.2. Isolated crude extract (0.59:2.9 g-eq) was added to 40
bM acetate buffer [pH 5. 2; 0. 4 M
2.5xlOO containing NaCQ; referred to as buffer 1αA] (!IM!) and equilibrated in advance with the same buffer.
ci Sepharose 6B column and eluted with buffer A. 5.5 each at a flow rate of 12 jll2/hour
Two samples were collected and the protein content was monitored. Fractions corresponding to each of the four peaks, designated fractions 1, 2, 3, and 4, were collected, dialyzed against distilled water, and lyophilized (2.99-eq each). A portion of fraction 3 (70m9: 1.5g-eq) was added to buffer A (
1.8x dissolved in 11IQ> and equilibrated with the same buffer.
It was applied to a Sephadex G-75 column of lOOcx and eluted with buffer A. 5. each at a flow rate of 10 iff/hour. I collected Ojl&. Fractions corresponding to each of the four peaks, designated fractions A, BSC, and D, were collected, dialyzed against distilled water, and lyophilized (1
．． 5 9-eq).

(Hereinafter, blank space) Process diagram 2 Extraction of kidney beans and fractionation of the extract.Bunyu B as a component 3. Protein content Protein content was determined using the Bio-Rad protein assay [Bio-Rad La
boratories, Richmond. USA
], and the distribution of proteins in the column eluent was investigated by absorbance at 280 nm.

4. Electrophoresis analysis Electrophoresis was performed using the method of Laen+mi (Reference 8).
) using 7.5% polyacrylamide gels with and without SDS (l4, wxl3ci
xl3Cz). Electrophoretic separation is 0.025M
Using tris-glycine buffer (pH 8.3), the test was carried out for 4 hours at a constant current of 20 IIIA (working voltage: 80 to 200 V). Coomassie brilliant in 10% acetic acid
Proteins in the gel were stained using Blu 10-250. Glycoprotein-containing bands were visualized using periodic acid and Schiff reagent (PAS) according to the method of Fairbanks et al. (Reference 9). The sample amounts used for protein staining and glycoprotein staining were 10-20 and 50 μ2, respectively.

Preparative scale polyacrylamide gel electrophoresis was performed using a 10% slab gel (2IIRXl3cz x
3 cm) at 25°C. The separated protein bands were separated using 2.5n+M} lysuglycine buffer (p
H8.3), were continuously collected by electrophoresis using a constant current of 30 mA.

5. Determination of Molecular Weight Molecular weight was determined by polyacrylamide gel electrophoresis (PAGE) using a 7.5% slab gel and a 50 m
It was determined by gel filtration on a Sephadex G-75 column (1.8xlOOcx) equilibrated with buffer A of M. The molecular weight marker used was bovine serum albumin (
6 8,0 0 0), egg albumin (45,000
), carbonic anhydrase (31,000), chymotrypsinogen A (25. O O O), soybean trypsin inhibitor (21,500) and horse heart cytochrome c (1
2.500).

6. Assay of Tribusin Inhibitory Activity Ability of trypsin to decompose N-a-penzoyl-DL-arginine-p-nitroanilide hydrochloride to generate p-nitroaniline having maximum absorption at 410 nm according to the method of Kakade (Reference 10) was measured in the presence and absence of sample.

7. Assay of α-amylase inhibitory activity α-amylase activity was measured by modifying Bernfeld's method (Reference 11). Amylase inhibitory activity was investigated by measuring amylase activity in the presence of the sample. The reaction mixture was as follows: appropriately diluted sample solution IfL (40 μQ) was added to 40 mM acetate buffer [pH 5.
4: In the presence of 10mM CaCI2t,
Dissolve pig pancreatic α-amylase at 25°C for 10 minutes? rJ
Pre-incubated with L (100μe) and then starch (2% soluble iffllooμQ) was added. 2
After incubating for 15 minutes at 5°C, the reaction was stopped by adding dinitrosalicylic acid reagent (200 μQ) followed by boiling for 5 minutes and cooling. Distilled water (2' ffj) was added to this solution, mixed, and left at room temperature for 15 minutes. Absorption at 540r+n+ was measured.

8. Measurement of Isoelectric Point 11 The isoelectric point was measured by a modified method of Aglund's method (Reference 12). l%pharmalyte pH
A linear sucrose gradient containing 3 to 1 O (Pharmacia) was formed into a 11Qxl2LKB column electrophoresis apparatus. Electrophoresis at a constant voltage of 400 V
Time went. Fractions (2 ml!) were collected and analyzed for inhibitory activity and pH.

Figure 1 shows the dose-response curve of the crude extract for the first instar larvae of the red bean weevil;
．． Doses of 29-eq or higher resulted in 100% mortality. At doses of 0.1 and 0.05 y-eq of kidney beans, the mortality rate was 70-80% and 30-80%, respectively.
It was 40%. L.D.

The value was approximately 0.069-eq. Based on these data, 0.25 and Q. A dose of 5 g-eq was chosen for bioassay. The precipitate (residue) obtained during the preparation of the crude extract gave a certain level of activity, probably due to contamination of the supernatant fraction (Table 1).

Growth inhibitory activity fraction of crude extract and residue
Mortality rate % crude extract
100 residue 25 control 7 total crude extract with Se
Fractionation was performed using PHAROSE 6B column chromatography. As shown in Figure 2, the elution profile based on protein content mainly consisted of four peaks. Except for the first peak at the void volume (which may be a macromolecule), the second, third, and fourth peaks are not clearly separated, and the appropriately divided fractions 1 to 4 are 0.
．． Doses of 259-eQ gave mortality rates of 0, 5, 100, and 0%, respectively (Table 2). Dose 05 9-
Fraction 3 was found to contain the most active fraction even when increasing eq.

Spring 2 Growth inhibitory activity fractions of fractions 1 to 4 obtained from crude bean extract by 1G Sepharose 6B' full filtration chromatography 9-eq Mortality rate % Yield (immediate) +
0.25 0 4712
0.25 5 19330.25
100 1364 0.25 0
1241 0.5 0 2 0.5 20 3 0.5 100 4 0.5 0 Trypsin inhibitory activity was observed in fractions 3 and 4. However, when active fraction 3 was further fractionated on a Sephadex G-75 column, the elution profiles based on protein and tribucin inhibitory activities did not overlap (Figure 3).

Based on the indicated protein content, fractionation was performed based on the four peaks and bioassay was performed (Table 3).

Fraction B, which gave 100% lethality, contained no trypsin inhibitory activity, while fractions A, C and D, which contained relatively high trypsin inhibitory activity, were virtually non-lethal. The elution profile based on α-amylase inhibitory activity was consistent with fraction B.

Brush 3fi Sephadex G-75 Growth inhibitory activity fractions of fractions A-D obtained from fraction 3 by gel filtration chromatography 9-eQ Mortality rate %A
0.25 0B O.
25 100G O. 25
0D 0.25 1
A O,5 B 0.5 C 0.5 D 0.5 Analysis of the components of fractions A, B, C and D by gel electrophoresis is shown in FIG. Fraction B, ~B, which constitutes fraction B
．． (B, and B, shown) gave almost a single band by protein and carbohydrate staining, indicating that this band was a glycoprotein.

When fraction B is separated by preparative electrophoresis,
A single band was obtained identical to that shown by analytical electrophoresis. Fractions A, C, and D, which contain trypsin inhibitory activity but not growth inhibitory activity, gave numerous bands on PAGE. By comparison with molecular weight markers, the molecular weight of fraction B was found to be slightly over 45,000, as shown in FIG.

In addition, the molecular weight of fraction B was determined using Sephadex, which was calibrated.
It was determined to be about 48,000 by gel filtration using a G-75 column (Figure 5). The isoelectric point of this inhibitor was found to be pH 4.46 (Figure 6).

As described above, the present inventors have isolated a chemical substance present in kidney bean seeds that is toxic to the growth of larvae of adzuki bean weevil and Japanese bean weevil, but not to humans. Molecular weight is approximately 48.000
It was identified as sugar hydroxide with an isoelectric point of pH 4.46. This substance exhibits α-amylase inhibitory activity, but
It was neither a phytohemagglutinin (lectin) nor a tribucin inhibitor. The present invention utilizes substances that play an important role in the self-defense mechanisms of kidney bean seeds to render commercially important beans resistant to breeding beetles.

Reference 1. S. Ishii: Bul1, Nat.
lnst. Agr. Sci. I(C), 185
(1952) 2. D. H. Janzen. I1, B. Juste
r&I. E. Liener:Science 19
2, 795 (1976) 3. A. M. R.
Gatehouse, J. ^． Gatehouse,
P. Dobie, A. M. Kilminster
&D. Boulter: J. Sci. F
oodAgric. 30, 948 (1979) 4.
K. Tanaka, H. Honda & 1, Yam
amoto: unpublished (1981) 5
．． J. R. Powers & J. D. Culb
ertson: J. Food Protection
45. 655 (1982)6. A. M. R.
Gatehouse, K. A. Fenton,
1, Jepson & D. J. Pavey: J
．． Sci. Food Agric. 37, 7
27 (1986) 7. M. Ishimoto and K. K
itamura: Japan J. Breed 3
8, 367 (198g)8. U. K. Laem
mli; Nature 227, 680 (19
70)9. G. Fairbanks, T.
L. Steak & D. F. II. Wall
aceBiochemistry 10. 2606
(1971) 10. M. L. Kakade+N
．． Simons & I. E. Liener:Cere
al Chet46. 518 (1969) 11.
R. Bernfeld: “Methods in
Enzymology, vol. 1, 1, C. S
．． P. Colowick & N. 0. Kap
lan ed. +p. 149, Axademic
Press, New York. 19551
2. Haglund:

[Brief explanation of drawings]

FIG. 1 is a graph showing the dose-response curve of crude extract for first instar larvae of Azuki bean weevil; FIG.
FIG. 3 is a graph showing the profile of gel filtration of crude kidney bean extract using a column; FIG.
H5. 2) is a graph showing the gel filtration profile of fraction 3 using a Sephadex G-75 column, and Figure 4 shows the results of PAGE of fractions A-D obtained by Sephadex G-75 chromatography. FIG. 5 is a graph of the molecular weight measurement of a growth-inhibiting substance (fraction B) using a gel filtration column calibrated with several molecular weight markers, and FIG. 6 is a graph showing the isoelectric properties of the growth-inhibiting substance. It is a graph of point measurements.

Claims (1)

[Claims] 1. Extracted from kidney bean seeds, with a molecular weight of about 48.0
00 and an isoelectric point of pH 4.46, which is a glycoprotein that inhibits the growth of adzuki bean weevil and Japanese bean weevil larvae. 2. A method for producing a growth inhibiting substance according to claim 1, comprising: (a) extracting kidney bean seed powder with a phosphate buffer; and (b) separating the extract by gel filtration chromatography using cross-linked agarose as a carrier. (c) further fractionating one of the fractions by gel filtration chromatography using cross-linked dextran as a carrier. 3. A growth inhibitor for adzuki bean weevil and Japanese bean weevil larvae, which contains the growth inhibitory substance according to claim 1 as an active ingredient.