JPH11255795A - Azodilyxofuranoside adenine derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new derivative comprising a specific azodilyxofuranoside adenine derivative, capable of raising the absorption percentage of phosphorus, or the like, in plants and manifesting growth promoting actions on mycorrhizal fungi for enhancing disease resistance and useful as an active ingredient, or the like, of a growth promoter for the mycorrhizal fungi. SOLUTION: This new azodilyxofuranoside adenine derivative is represented by the formula and is capable of manifesting growth promoting actions on mycorrhizal fungi having effects on an improvement in the absorption percentage of phosphorus, or the like, in plants and growth of the plants and enhancement of resisting power to diseases and useful as an active ingredient, or the like, of a growth promoter for the mycorrhizal fungi. The compound is obtained by cutting dried Laminaria japonica, dipping the cut Laminaria japonica in a 75% methyl alcohol, extracting the Laminaria japonica, vacuum concentrating the extract solution, fractionating the resultant concentrate by a flash chromatography using a silica-based adsorbent resin-packed column, further treating the resultant fraction with a gel chromatography using a porous cellulose-packed column and fractionating the resultant fraction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel azodyloxofuranosidoadenine derivative having a growth promoting action on mycorrhizal fungi.

[0002]

2. Description of the Related Art Mycorrhiza of plants is formed of fungi having a symbiotic relationship in which symbiotic bacteria collect moisture and nutrients from soil and supply them instead of absorbing carbohydrates from plants. Such mycorrhizas include ectomycorrhizal fungi, in which the mycelia wraps the surface of the fine roots and the outside of the cells, and endophytic mycorrhizas, in which the mycelium enters the root cells. Mycorrhizal fungi that enter the root cells to form sacs (Vesicule) and dendrites (Arbuscule) are called VA mycorrhizas.

[0003] VA mycorrhizal fungi belong to zygomycetes, and particularly play an important role in the absorption of phosphorus and potassium. Therefore, the rate of absorption of phosphorus and the like in plants in which VA mycorrhiza is formed is improved, and the growth of plants is improved. It is said that resistance to disease will increase.

[0004] The only known compound capable of promoting the growth of mycorrhizal fungi is eupatiline (Ishii et al., 1997) purified from an annual herb extract of bahiagrass, which is a flavonoid-based compound. There are no other known compounds that have the same effect.

[0005]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an azodilyxofuranoside adenine derivative which is a novel compound having an activity of promoting the growth of mycorrhizal fungi.

In the present invention, as a result of conducting an extraction test of substances related to the growth-promoting action of mycorrhizal fungi from various plants, the azodilixofuranosidoadenine derivative represented by the following formula (2) was excellent. They have been found to exhibit the growth promoting action of mycorrhizal fungi.

[0007]

Embedded image

In the present invention, the compound [Chemical Formula 2] is produced by preparative chromatography of an alcoholic solvent of a marine algae using a column packed with a silica-based adsorption resin and a column packed with porous cellulose. The desired fraction is fractionated by column chromatography using a solvent containing acetonitrile on a column packed with a silica-based adsorption resin to obtain a further fraction. The inventors of the present application have found that the fractionated substance showing a predetermined peak in column chromatography is an azodilyxofuranoside adenine derivative represented by Chemical formula 2, which shows a growth promoting action of mycorrhizal fungi. I found it.

[0009] Marine algae as raw materials for crude extracts include:
As is clear from the examples described later, Macomb (Lami
kelp (Nambu: Laminari) such as naria japonica)
In addition to a), marine algae such as seaweed (Undaria) such as seaweed (Undaria pinnaltifida) have been used to obtain favorable results.

As an actual procedure of the preparative chromatography, first, a crude alcohol extract of marine algae is fractionated by flash chromatography using a column packed with a silica-based adsorption resin, and the fraction is further subjected to porosity separation. Fractionation is performed by gel chromatography using a column packed with a functional cellulose.

The above fractionation method will be described in detail. The methanol extract of marine algae is fractionated on a column packed with octadecyl silica by flash chromatography using a solvent having a methanol concentration of 0% to 25%. %, And then fractionated by gel chromatography with a methanol solvent using porous cellulose as a carrier, and the fraction corresponding to the third peak appearing in the order of elapsed time is collected. .

The fraction is then subjected to column chromatography using a column packed with a silica-based adsorption resin such as octadecyl silica, so as to linearly increase the concentration gradient of acetonitrile in the solvent over time from 0% to 30% or more. Fractionation is performed by chromatography, and the fraction having the largest peak (the one having the maximum ultraviolet absorption at 254 nm) is collected. If necessary, a predetermined column chromatography operation or the like can be repeated to obtain a purified product with higher purity.

The physicochemical properties of the compound represented by the formula (I) obtained by the above-mentioned operation (described later in detail as an example) are as follows. In addition, the measured value was measured using the following equipment.

Specific rotation: DIP-100 manufactured by JASCO Corporation
0, Infrared absorption spectrum: Infrared Spect manufactured by Hitachi, Ltd.
or Meter 270-30, mass spectrometry: JMS-DX303HF, manufactured by JEOL, NMR spectrum: JNM-EX400, manufactured by JEOL Co., Ltd. [Physical and chemical properties] Property: oil Specific rotation: [α] _D + 31 ° (C0.2, CH ₃ O
H) Molecular weight: 556 Molecular formula: C ₂₂ H ₂₈ O ₆ N ₁₂ Mass spec: 557 [M + H] ⁺ , 579 [M + Na]
⁺⁺ Infrared absorption spectrum (KBr method) (ν c
m ^-1 ): 3700-3000, 2900-2800, 1680, 1610, 1200, 1135 1H-nuclear magnetic resonance spectrum: (400 MHz, CD
₃ OD solution): δ ppm (integral, multiplicity, coupling constant) 2.68 (3H, s), 2.68 (3H, s), 3.20 (1H, dd, 12.5,2.5), 3.35
(1H, dd, 9.5,6.9), 3.36 (1H, dd, 9.5,3.7), 3.45 (1H, dd, 1
2.5,10.5), 4.38 (1H, dd, 4.7,5.3), 4.40 (1H, dd, 4.7,5.
3), 4.47 (1H, ddd, 10.3,4.7,2.5), 4.52 (1H, ddd, 6.9,4.7,
3.7), 4.76 (1H, dd, 4.7,5.3), 4.77 (1H, dd, 4.8,5.3), 6.0
3 (1H, d, 4.7), 6.04 (1H, d, 4.8), 8.40 (1H, s), 8.40 (1H,
s), 8.48 (1H, s), 8.49 (1H, s) 13 C-NMR spectrum: (100 MHz, CD
₃ OD solution): δ ppm (multiplicity) 39.2 (q), 40.8 (q), 57.3 (t), 57.3 (t), 75.6 (d), 75.6
(d), 75.7 (d), 75.8 (d), 80.7 (d), 80.9 (d), 92.0 (d), 9
2.1 (d), 122.0 (s), 122.0 (s), 144.9 (d), 145.0 (d), 14
6.9 (d), 147.0 (d), 150.7 (s), 150.7 (s), 153.0 (s), 15
3.0 (s).

[0015]

Example: Dried makombu (Laminaria japonica)
1 kg was cut into 5 cm pieces, immersed in 75% methyl alcohol (hereinafter abbreviated as MeOH), and after 24 hours, a 75% MeOH solution was recovered. This solution was concentrated under reduced pressure by an evaporator to obtain 1 liter of a concentrated solution.

This concentrated solution is used as an O.C.
It was introduced into a DS DM1020T column (diameter φ: 20 mm, length: 250 mm, dry volume: 3 liters),
Flash chromatography was performed eluting stepwise with 5 l of 0% MeOH solution and 5 l of 25% MeOH solution.

The obtained 25% MeOH eluate was concentrated under reduced pressure using an evaporator, and the eluate was subjected to gel chromatography under the following conditions. The obtained chromatogram is shown in FIG.

That is, porous cellulose (Cellulofin)
e GCL-25sf) using a column (diameter φ25 mm, length 300 mm) packed with GCL-25sf) to elute 7.5% ethyl alcohol (hereinafter abbreviated as EtOH) at a flow rate of 2.5 ml / min. Was performed. And
Peak 3 in FIG. 1 (between 60 minutes and 72 minutes after the start of elution)
Eluate (fraction) was collected.

A column packed with ODS (octadecylsilane) under high pressure (COSMOSIL manufactured by Nakarai Co., Ltd.)
Packed Column / ODS5C18-AR: Diameter φ10m
m, length 250 mm) and column chromatography was performed using ultraviolet absorbance at a wavelength of 254 nm (range 0.32), and FIG. 2 shown in the profile shown in FIG.
0.4 mg of an eluate corresponding to the peak 1 was obtained.

In addition, this column chromatography is
The starting solvent was 0.1% trifluoroacetic acid (TFA),
The final solvent was a mixed solution of 0.1% TFA and 30% acetonitrile, which was supplied to the ODS column at a flow rate of 1.8 ml / min, and the concentration gradient of acetonitrile with time was linearly changed from 0% to 30%. Increase to mold (50 minutes)
(Linear gradient).

The physicochemical properties of 0.4 mg of the eluate (fraction) corresponding to peak 1 in FIG. 2 are as described in (1) above, and this is the compound represented by Chemical Formula (2) above. It was confirmed.

The effects of the obtained fractions on the growth of hyphae of VA mycorrhizal fungi were evaluated by the following method.

That is, 10 ml of a 1.5% elementary agar medium (using a petri dish with a diameter of 70 mm) to which 0.4 mg of the above fraction (equivalent to 10 g of dried makombu) was added at 121 ° C.
After that, 4 spores of Gigaspora margarita (surface-disinfected) were placed in a petri dish, and incubated at 30 ° C. in the dark. Two weeks later, hyphal elongation from the spores was measured by a stereoscopic microscope equipped with a CCD camera and an image processing method using a personal computer (Ishii et al., 1996). The results are shown in Table 1. A blank test for incubating spores of Gigaspora margarita was performed in exactly the same manner except that only distilled water was added to the plain agar medium as a control, and the results are shown in Table 1.

[0024]

[Table 1]

As is evident from the results in Table 1, all fractions comprising the compound represented by the chemical formula (2) show a growth promoting action of VA mycorrhizal fungi, and markedly increase mycelial growth as compared with the blank. Admitted.

[0026]

As described above, the present invention relates to an azodilixofuranosidoadenine derivative represented by the above-mentioned formula (2), and this compound has an excellent action to promote the growth of mycorrhizal fungi. Therefore, there is an advantage that a novel chemical substance having excellent properties can be provided as an active ingredient of a growth promoting agent for mycorrhizal fungi.

[Brief description of the drawings]

FIG. 1 is a chart showing the relationship between the elapsed time of chromatography using a porous cellulose packed column and ultraviolet absorbance.

FIG. 2 is a table showing the relationship between the elapsed time of chromatography using an ODS packed column and ultraviolet absorbance.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Takaaki Ishii 4-1-27 Oguri, Matsuyama-shi

Claims (1)

[Claims] An azodilyxofuranosidoadenine derivative represented by the following formula: Embedded image