JPH0517768A - New antimicrobial and oxidation-resistant substances, ysl-8 and ysl-9, production thereof and new antimicrobial and antioxidizing agent - Google Patents

Abstract

PURPOSE:To obtain a new antimicrobial and oxidation-resistant substance useful as germicide agriculture and horticulture, preservative for food, etc., because of excellent antimicrobial properties and anti-oxidation activity thereof by extracting the substance from Aruncus dioicus var. tenuifolius of Rosaceae. CONSTITUTION:A new antimicrobial and oxidation-resistant substance YSL-8 having the following characteristics: Properties, colorless and oily; molecular formula, C19H18O7; molecular weight, 358; color reaction, positive to vanillin sulfate, iodine reaction, etc.; solubility, soluble in methanol, acetone, etc., and hardly soluble in hexane, water, etc. The substance is obtained by extracting a plant body of Aruncus dioicus var. tenuifolius of Rosaceae with a solvent such as water, ethanol or acetone and then isolating and purifying the substance from the extract by a method utilizing a difference in absorption affinity on silica gel, reverse phase silica gel, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to novel antibacterial and antioxidant active substances YSL-8 and YSL-9, a process for producing them and a novel antibacterial and antioxidant containing the substance as an active ingredient.

[0002]

2. Description of the Related Art Synthetic compounds are the mainstream of currently used agricultural and horticultural germicides or antioxidants used for the preservation of foods, and some of them are microbial metabolites such as antibiotics.

[0003]

[Problems to be Solved by the Invention] Plants have antibacterial activity, insecticidal activity or antioxidant activity in order to protect themselves from harmful active enzymes generated by invasion of pathogenic bacteria and pests, or by receiving ultraviolet rays. The substance is produced and secreted to maintain the individual. The present invention seeks out and elucidates such a protective substance of the plant itself, and a novel antibacterial and antioxidative active substance that can be used as an agricultural and horticultural fungicide or a food preservative, etc. and a novel antibacterial substance containing the substance as an active ingredient. It is intended to provide an antioxidant.

[0004]

Means for Solving the Problems The present inventors collected a wide variety of wild substances having strong resistance to pests and screened each plant for the physiological activity of the extract. As a result, it has excellent antibacterial and antioxidant activities from the extract of Yamabuki-shouma (Rosaceae),
We have succeeded in isolating two novel substances with very similar physicochemical properties, and completed the present invention. These two novel antibacterial and antioxidant active substances have been developed by the present inventors to use YSL-
8 and YSL-9, which have the following physicochemical properties. [1] YSL-8 (1) Property: colorless oil (2) Molecular formula: C ₁₉ H ₁₈ O ₇ (3) Molecular weight: 358 (4) Ultraviolet absorption spectrum (in methyl alcohol) λmaxnm (ε): 205 (11990) , 217 (13010), 248 (sh.6030),
302 (sh.7520), 332 (10200). (5) Infrared absorption spectrum (potassium bromide tablet method) (Fig. 1) cm ^-1 : 3360, 2960, 1760, 1740, 1710, 1695, 1685, 16
35, 1600, 1535, 1515, 1447, 1360, 1250, 1200, 1160,
1120, 1050, 1005, 980, 920,860, 815, 760, 600. (6) ¹ H-NMR spectrum (270MHz, in heavy acetone) (Fig. 2) δ: 1.93 (3H, s), 4.66 (2H, brs) , 4.80 (1H, brs), 5.1
4 (2H, brs), 5.19 (1H, brs), 5.60 (1H, d, J = 4.3), 6.39
(1H, d, J = 15.8), 6.98 (1H, d, J = 8.3), 7.05 (1H, t, J =
4.0), 7.13 (1H, d, J = 8.3), 7.26 (1H, d, J = 1.7), 7.66
(1H, d, J = 15.8). (7) ¹³ C-NMR spectrum (67MHz, in heavy acetone) (Fig. 3) δ (OFR): 19.13 (q), 60.71 (t), 68.52 (d), 78.30 (d), 8
4.86 (d), 114.26 (d), 115.12 (t), 115.12 (d), 116.21
(d), 122.72 (d), 127.03 (s), 129.91 (s), 140.38 (s), 14
4.64 (d), 145.67 (s), 146.73 (d), 148.47 (s), 166.20
(s), 170.12 (s). (8) Mass spectrum (EI-MS) m / z: 358 (M ⁺ ), 310, 296, 278, 272, 262, 236, 180, 17
8, 163, 150, 136 (B), 121, 107, 89, 77. (9) Color reaction: positive for vanillin sulfate, phosphomolybdic acid and iodine reaction.

(10) Solubility: methanol, ethanol,
Soluble in acetone and ethyl acetate. Insoluble in hexane, chloroform and water. [2] YSL-9 (1) Property: colorless oil (2) Molecular formula: C ₁₉ H ₁₈ O ₇ (3) Molecular weight: 358 (4) Ultraviolet absorption spectrum (in methyl alcohol) λmaxnm (ε): 205 (12550) , 216 (13120), 245 (sh.6800),
300 (sh.7520), 331 (10110). (5) Infrared absorption spectrum (potassium bromide tablet method) (Fig. 1) cm ^-1 : 3350, 2925, 1760, 1745, 1710, 1695, 1685, 16
35, 1600, 1515,1450, 1370, 1350, 1280, 1260, 1180,
1155, 1118, 1005, 922, 855,820, 760, 600. (6) ¹ H-NMR spectrum (270 MHz, in heavy acetone) (Fig. 2) δ: 1.92 (3H, s), 4.77 (1H, brs), 4.87 (2H, brs), 4.8
7 (1H, brs), 5.14 (1H, brs), 5.21 (1H, brs), 5.62 (1H,
brs), 6.41 (1H, d, J = 15.8), 6.83 (1H, brs), 6.98 (1H,
d, J = 7.9), 7.12 (1H, d, J = 7.9), 7.27 (1H, s), 7.68 (1H,
(7) ¹³ C-NMR spectrum (67MHz, in heavy acetone) (Fig. 3) δ (OFR): 19.00 (q), 58.89 (t), 70.59 (d), 75.96 (d). , 8
5.09 (d), 114.15 (d), 115.05 (d), 115.05 (t), 116.14
(d), 122.74 (d), 126.91 (s), 129.15 (s), 140.06 (s), 14
5.41 (s), 146.71 (d), 147.66 (d), 148.27 (s), 166.31
(s), 168.88 (s). (8) Mass spectrum (EI-MS) m / z: 358 (M ⁺ ), 329, 281, 239, 224, 207, 180, 178, 16
3, 150, 136 (B), 121, 107, 89, 82, 77. (9) Color reaction: positive for vanillin sulfate, phosphomolybdic acid and iodine reaction.

(10) Solubility: methanol, ethanol,
Soluble in acetone and ethyl acetate. Insoluble in hexane, chloroform and water. Table 1 shows Rf values of thin-layer chromatography of YSL compounds with various developing agents. The detection was performed by spraying vanillin sulfuric acid and then heating it on a hot plate.

[0007]

[Table 1]

Developing agent A: chloroform: methanol = 9: 1 (v / v) B: ethyl acetate: methanol = 10: 0.5 (v / v) C: methanol: water = 7: 3 (v / v) D: Acetonitrile: Water = 6: 4 (v / v) Thin layer: A, B: Silica gel 60F254 plate (Merck, No.5715) C, D: RP-8F254 plate (Merck, No.15388) YSL compound Is a wildflower belonging to the family Rosaceae
( Aruncu s d ioicus v. Tenuifoli s) plant is produced by extracting with a solvent and refining. As above,
Examples include water, ethanol, acetone and the like. Y of the present invention
For isolation and purification of SL compounds, any method such as silica gel, reverse layer silica gel, Diaion HP-20 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) or the like using a difference in adsorption affinity or a method using difference in solubility between solvents is used. , Isolated respectively,
Alternatively, they can be used in appropriate combination or repeated. The active portion can be confirmed by silica gel thin layer chromatography (TLC).

To use the YSL-8.9 of the present invention as an agricultural and horticultural fungicide, the YSL-8.9 of the present invention and a suitable carrier and auxiliary agent such as a surfactant, a binder and a stabilizer can be used. And may be blended by a conventional method into a wettable powder, an emulsion, a liquid agent, a sol agent (flowable agent), an oil agent, a powder agent, a DL (driftless type) powder agent, a fine granule, a granule and the like. The content of these preparations is in the range of 0.1 to 90% (same as below by weight) for wettable powders, emulsions, sol preparations, oil preparations, and liquid preparations, and for powder preparations, DL powder preparations, fine granule preparations, and coarse powder preparations. Is 0.1 to 3
%, And in the case of granules, it can be compounded in the range of 0.1 to 10%.

In the above, usable carriers are as follows:
As long as it is commonly used for agricultural and horticultural chemicals, it can be used as a solid or a liquid and is not limited to a particular one. For example, as the solid carrier, mineral powder (kaolin, bentonite, clay, montmorillonite, talc, vermiculite, gypsum, calcium carbonate, white carbon, silica sand, ammonium sulfate, urea, etc.), vegetable powder (starch, crystalline cellulose, etc.), Examples thereof include polymer compounds (petroleum resin, etc.), alumina, silicates, sugar polymers and the like.

As the liquid carrier, water, alcohols (methanol, ethanol, n-propanol, ethylene glycol, glycerin, etc.), aromatic hydrocarbons (xylene, methylnaphthalene, etc.), halogenated carbons (dichloromethane, etc.) are used. , Ethers (tetrahydrofuran etc.), ketones (methyl ethyl ketone, cyclohexanone etc.), esters, nitriles, sulfoxides (dimethyl sulfoxide etc.), alcohol ethers (ethylene glycol monomethyl ether, ethylene glycol monoethyl ether etc.), aliphatic Or, alicyclic hydrocarbons (such as cyclohexane), industrial gasoline (such as solvent naphtha), and petroleum fractions can be mentioned.

In addition, in the preparation of emulsions, wettable powders, flowable agents (sols), etc., emulsification, dispersion, solubilization, wetting,
Surfactants (or emulsifiers) are used for the purposes of foaming, lubrication, spreading and the like. Such surfactants include nonionic type (polyoxyethylene alkyl ether, polyoxyethylene alkyl ester, polyoxyethylene sorbitan alkyl ester, sorbitan alkyl ester, etc.), anionic type (alkylbenzene sulfonate, alkyl sulfosuccinate). , Alkyl sulphate, polyoxyethylene alkyl sulphate, aryl sulphonate etc.), cationic type (alkyl amines (lauryl amine, stearyl trimethyl ammonium chloride, alkyl dimethyl dibenzyl ammonium chloride etc.), polyoxyethylene alkyl amines etc.), Examples thereof include amphoteric types (carboxylic acid (betaine type), sulfate ester salts, etc.), but are not limited to these examples.

In addition to these, various auxiliary agents such as polyvinyl alcohol (PVA), carboxymethyl cellulose (CMC), gum arabic, polyvinyl acetate, gelatin, casein, sodium alginate, tragacanth gum, xanthan gum and the like can be used. Further, while the YSL compound of the present invention is used as an agricultural and horticultural germicide, it can be used as a mixture with other germicidal components, insecticidal components, acaricidal components, nematicidal components and the like.

By mixing them, the applicability (applicable diseases, method of use, time of use, etc.) can be expanded, and in some cases, synergistic control effect can be expected by the synergistic action of each utilization component. You can Also, Y
When the SL compound is used as an antioxidant, the compound may be used as it is, or may be appropriately mixed with a carrier for foods, cosmetics or pharmaceuticals or a diluent according to a conventional method to obtain a powder,
It can be in the form of granules, emulsions, solutions, suspensions and the like.

The amount of the fungicide for agricultural and horticultural use of the present invention generally varies depending on various factors such as the target disease, its occurrence state, weather, dosage form, application method, place of application and time of application, but it is generally powder or granules. The dosage is 0.5 kg to 25 kg per 10 ares. In the case of a formulation that is finally used in a liquid state such as an emulsion or a wettable powder, the active ingredient concentration in the formulation is generally 2.5 g per 10 are when diluted with 100 to 5000 L of water and then sprayed. It may be prepared so that the applied amount of the active ingredient is 125 g.

(Test Example) Test Example 1 (Antibacterial Activity Test) The antibacterial activity against various bacteria was examined using the paper disk method. The paper disk was immersed in a 1000 ppm acetone solution of the YSL-8 and -9 compounds, air-dried, and then assayed. The results are shown in Table 2.

[0017]

[Table 2]

Test Example 2 (Antioxidant activity test) An antioxidative test was carried out by the iron thiocyanate method (Agric. Biol. Che.
m., 47 (3) 521-528, 1983). 99.5%
Dissolved in ethanol, 0.01% sample solution 1 ml, 99.5%
1 ml of 2.51% linolenic acid solution dissolved in ethanol, 0.05
% Phosphate buffer (pH 7.0) 2 ml and distilled water 1 ml vial with screw cap (φ = 24, h = 40 mm)
And left in the dark at 40 ° C. in the open. After a certain time, this sample solution was placed in a 0.1 ml test tube, and 9.7 ml of 75% ethanol and 30% ammonium thiocyanate solution were added to 0.1 ml.
Add 1 ml. 3.5% of 2 × 10 ^-2 M ferrous chloride in this solution
Add 0.1 ml of hydrochloric acid solution to develop a red color. 3 minutes after the addition of ferrous chloride, the degree of color development was colorimetrically determined by UV absorption at 500 nm. The results are shown in Fig. 4.

As is clear from FIG. 4, the novel antibacterial and antioxidative active substances YSL-8 and YSL-9 of the present invention are clearly superior to tocopherol used as a food preservative and almost comparable to the synthetic antioxidant BHT. It showed high antioxidative activity.

[0020]

EXAMPLES The present invention will be specifically described below with reference to examples. However, the technical scope of the present invention is not limited by these examples. Example 1 2.7 kg of fresh yamabuki leaf, which was collected in a primeval forest around Lake Shikotsu in Chitose City, Hokkaido, was mechanically treated by hitting with wood rod and then extracted by immersion with 20 L of deionized water.
The extraction was carried out 3 times with the water exchanged every day to obtain a total of 60 L of the plant extract.

Treatment of this extract with ethyl acetate yields YS
The L compound was redissolved in the ethyl acetate layer. The ethyl acetate extract was concentrated to dryness under reduced pressure to obtain 34.95 g of a crude extract.
The crude extract was again dissolved in 2 L of ethyl acetate, and the ethyl acetate solution was treated with saturated aqueous sodium hydrogen carbonate and then with 1/50 N hydrochloric acid to obtain an ethyl acetate-soluble neutral fraction. This fraction was dissolved in a solvent of n-hexane-ethyl acetate (1: 1), and silica gel (silica gel 60, No.7734, manufactured by Merck).
The column was fed at the top of 200 ml. After washing the column with 500 ml of n-hexane-ethyl acetate (1: 1), the active substance was completely eluted by flushing with 500 ml of ethyl acetate. This fraction was dissolved in a solvent of n-hexane-ethyl acetate (2: 8), passed through a silica gel column again, and the column was washed with 500 ml of this solvent.

The flow-through fraction thus obtained was dissolved in a small amount of ethyl acetate and adsorbed on a reverse layer of silica gel (Silica gel 60 silanized, manufactured by Merck), and ethyl acetate was removed under reduced pressure. This sample-adsorbed silica gel was supplied to the upper end of 200 ml of a reverse-phase silica gel column previously packed with water. Elution was performed using 500 ml of each while increasing the methanol concentration with respect to water by 5%.

The active substance was eluted in the 20% to 30% methanol elution fraction. This fraction was purified again by silica gel column chromatography on the reverse layer using n-propanol-water as a solvent system. N-propanol concentration of developing solvent is 15,17.5,
1000 ml of each was used in 3 steps of 20%. When 250 ml each was collected, the active substance was eluted in the 5th to 8th fractions. This was applied to a column of Sephadex LH-20 (φ32 × 410 mm) using CHCl ₃ -EtOAc-MeOH. (4.5: 4.5: 1) as a solvent system, and each 10 g of the active substance was separated and eluted. . Pure Y from fractions 34-36
425.8 mg of SL-8 could be isolated. Fraction number
388.1 mg of YSL-9 was successfully isolated by performing column chromatography of Sephadex LH-20 again under the same conditions on the 37-50 fractions.

Next, an example of using YSL-8, 9 as a fungicide for agriculture and horticulture will be described. "Parts" in the examples
Indicates parts by weight. Example 2 Wettable powder YSL-8 or 95 5 parts, kaolin 57 parts, diatomaceous earth
35 parts, polyoxyethylene alkyl aryl ether 1.
5 parts and 1.5 parts of sodium lignin sulfonate are uniformly mixed to obtain a wettable powder. Example 3 0.5 parts of powder YSL-8 or 9 and 99.5 parts of talc are uniformly pulverized and mixed to obtain a powder. Example 4 Granules YSL-8 or 9 3 parts, sodium lignin sulfonate
Add 22 parts of water to 3.5 parts of clay and 93.5 parts of clay, knead, granulate, mold and dry to obtain granules.

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, a novel antibacterial and antioxidant active substance YSL which is useful as an agricultural / horticultural germicide and a preservative for foods.
-8 and YSL-9 and their methods of manufacture were provided.

[Brief description of drawings]

FIG. 1 is a diagram showing infrared absorption spectra of YSL-8 and YSL-9 of the present invention.

FIG. 2 is a chart showing ¹ H-NMR spectrum of the present inventions YSL-8 and YSL-9.

FIG. 3 ¹³ C-NMR of the present invention YSL-8 and YSL-9.
It is a figure which shows a spectrum.

FIG. 4 is a diagram showing the results of an antioxidant activity test of YSL-8 and YSL-9 of the present invention.

Claims (4)

[Claims] 1. A novel antibacterial and antioxidant active substance YSL-8 having the following physicochemical properties. Property: Colorless oil Molecular formula: C ₁₉ H ₁₈ O ₇ Molecular weight: 358 Ultraviolet absorption spectrum (in methyl alcohol) λmaxnm (ε): 205 (11990), 217 (13010), 248 (sh.6030),
302 (sh.7520), 332 (10200). Infrared absorption spectrum (potassium bromide tablet method) cm ^-1 : 3360, 2960, 1760, 1740, 1710, 1695, 1685, 16
35, 1600, 1535, 1515, 1447, 1360, 1250, 1200, 1160,
1120, 1050, 1005, 980, 920,860, 815, 760, 600. ¹ H-NMR spectrum (270MHz, in heavy acetone) δ: 1.93 (3H, s), 4.66 (2H, brs), 4.80 (1H, brs) , 5.1
4 (2H, brs), 5.19 (1H, brs), 5.60 (1H, d, J = 4.3), 6.39
(1H, d, J = 15.8), 6.98 (1H, d, J = 8.3), 7.05 (1H, t, J =
4.0), 7.13 (1H, d, J = 8.3), 7.26 (1H, d, J = 1.7), 7.66
(1H, d, J = 15.8). ¹³ C-NMR spectrum (67MHz, in heavy acetone) δ (OFR): 19.13 (q), 60.71 (t), 68.52 (d), 76.30 (d), 8
4.86 (d), 114.26 (d), 115.12 (t), 115.12 (d), 116.21
(d), 122.72 (d), 127.03 (s), 129.91 (s), 140.38 (s), 14
4.64 (d), 145.67 (s), 146.73 (d), 148.47 (s), 166.20
(s), 170.12 (s). 2. A novel antibacterial and antioxidant active substance YSL-9 having the following physicochemical properties. Property: Colorless oil Molecular formula: C ₁₉ H ₁₈ O ₇ Molecular weight: 358 Ultraviolet absorption spectrum (in methyl alcohol) λmaxnm (ε): 205 (12550), 216 (13120), 245 (sh.6800),
300 (sh.7520), 331 (10110). Infrared absorption spectrum (potassium bromide tablet method) cm ^-1 : 3350, 2925, 1760, 1745, 1710, 1695, 1685, 16
35, 1600, 1515,1450, 1370, 1350, 1280, 1260, 1180,
1155, 1118, 1005, 922, 855,820, 760, 600. 1 H-NMR spectrum (270 MHz, in deuterated acetone) δ: 1.92 (3H, s ), 4.77 (1H, brs), 4.87 (2H, brs), 4.8
7 (1H, brs), 5.14 (1H, brs), 5.21 (1H, brs), 5.62 (1H,
brs), 6.41 (1H, d, J = 15.8), 6.83 (1H, brs), 6.98 (1H,
d, J = 7.9), 7.12 (1H, d, J = 7.9), 7.27 (1H, s), 7.68 (1H,
d, J = 15.8). ¹³ C-NMR spectrum (67MHz, in heavy acetone) δ (OFR): 19.00 (q), 58.89 (t), 70.59 (d), 75.96 (d), 8
5.09 (d), 114.15 (d), 115.05 (d), 115.05 (t), 116.14
(d), 122.74 (d), 126.91 (s), 129.15 (s), 140.06 (s), 14
5.41 (s), 146.71 (d), 147.66 (d), 148.27 (s), 166.31
(s), 168.88 (s). 3. A novel antibacterial / antioxidant substance characterized by extracting a plant of Yamabukishouma with a solvent, and separating and collecting the novel antibacterial / antioxidant substance according to claim 1 and / or 2 from the extract. Manufacturing method. 4. A novel antibacterial and antioxidant, which comprises YSL-8 according to claim 1 and / or YSL-9 according to claim 2 as an active ingredient.