KR100422178B1 - Composition for inhibiting mycotoxin production - Google Patents

Abstract

The present invention relates to a fungal toxin production inhibitor composition using a natural physiologically active substance, and more particularly, to a fungal toxin production inhibitor composition using a pepper extract.

Description

Composition for inhibiting mycotoxin production

The present invention relates to a fungal toxin production inhibitor composition using a natural physiologically active substance, and more particularly, to a fungal toxin production inhibitor composition using a pepper extract.

Plant diseases occurring during the cultivation of crops and economic losses caused by plant diseases during transportation or storage after harvesting amount to about 5 to 50%. In developing countries, losses are so severe that losses from phytopathogenic bacteria cannot be achieved without the use of pesticides. (Agrios, GN In Plant Pathology, 4th Ed., Academic Press, pp 25-37, 1997 .; Oreke, EC, Dehne, HW, Schonbeck, F., and Weber, A. Crop production and crop protection: Estimated Losses in Major Food and Cash Crops.Elsevier, Amsterdam, 1994.) Fujita investigated yields and shipments declines without pesticide use. Rice and varieties showed a 20-30% reduction, while apples and peaches did not. It could not be harvested, and other crops were reported to have caused enormous damage. The damage of plant diseases is known to be not only reduced in yield but also strongly toxic to humans and animals by secondary metabolites produced by pathogens. (Ames, BN Sci ., 204, 587-593, 1979 .; Vesconder, RF and Golinski, P. In Fusarium-mycotoxins, taxonomy and pathogenicity, ed. By J. Chelkowski, Amsterdam, Elsevier. Pp. 1-39, 1989.)

Recently, as one of the important factors causing direct and indirect adverse effects on the quality improvement and quality preservation of agricultural products, as well as developed countries, the problem of fungal toxin caused by phytopathogenic fungi has emerged seriously.

Ain phytopathogenic fungal secondary metabolites produced by the fungi Aspergillus flavus furanyl toxin B ₁ (aflatoxin B ₁₎ is a colorless, odorless, a fungus toxins. (Diner, UL, Cole, RJ, Sanders, TH, Payak, GA, Lee, LS, and Klich, MA Ann. Rev. phytopathol. , 25, 249-270, 1987) The structure and biosynthesis process are shown in FIG. It was.

Difuranocoumarin compounds associated with apuratoxin B ₁ are known to cause mutations, carcinogenesis and malformations in humans and animals. (Fugimoto, Y., Hampton, LL, Writh, PJ, Wang, NJ, Xie, JP, and Thorgenirsson, SS Cancer Res ., 54, 281-285, 1994 .; Hosono, S., Chou, MJ, Lee, CS, and Shin, C. Oncogene , 8, 491-496, 1993.) Contamination of agricultural products, especially nuts, is a major concern in many countries and a great deal of research is being done. Many studies have been reported on the environmental factors such as A. flavus , which produces toxic apuratoxin B ₁ , as well as the types of fungi, the biosynthetic processes of toxins, and the effects of toxins on humans and animals. . Aspergillus spp. Has yet to be studied about the different kinds of toxins produced by the world, and orchratoxin A produced by Aspergillus ochraceus is a representative example. (Abarca, ML, Bragular, MR, and Castella, G. J. Food Prot ., 60, 1580-1582, 1997.) Okratoxin A was known about 35 years ago in its chemical structure. (Merwe, KJ, van der, Steyn, PS and Fourie, L. J. Chem. Soc ., 7083-7088, 1965.) Currently crops and nuts (Scott, PM, Van-Waleek, W., Kennedy, B ., and Anyeti, D. J. Agric.Food Chem. , 20, 1103-1109, 1972; Krogh P., Hald, B., and Pederson, EJ Acta Pathol.Microbiol.Scand. (B) Microbiol.Imminol ., 81, 689-695, 1973.) and stored foods (Ucno, T. CRC Crit. Rev. Toxicol. , 14, 33-132,1985 .; Hohler, D. Z. Ernachrungswiss , 37, 2-12, 1998.), and is known to cause kidney disease, liver disease, malformation and immunosuppression in higher organisms (Cole, R. J ,. and Cox, RH Handbook of Toxic Fungal Metabolites, Academic Press). , 1981.)

Ocratoxin A has a structural characteristic in which dihydroisocumarin is bonded to L-phenylalanine in which a carboxyl group is substituted on a carbon adjacent to an amide group.

Treatment of okratoxin A with acid produces phenylalanine and isocumarin. As Stromer et al by reacting NADPH and oxygen with a Ochratoxin A and rat, pig and human liver microsomal (4 R) -4- hydroxy Ochratoxin A and (4 S) -4- hydroxy Ochratoxin A It was confirmed that the same two kinds of metabolites were produced, and using the liver of rabbits, 10-hydroxyocratoxin A was additionally produced in addition to the above two metabolites. The metabolism of okratoxin A in vivo is inhibited by carbon monoxide and metricaponecm, and its metabolism is elevated when using livers of animals pretreated by phenobarbital. Therefore, based on these findings, the metabolites are known to be metabolized by monooxygenase, which depends on cytochrome P450, but the biosynthetic pathways in vivo have not been elucidated.

Although many studies have been carried out at home and abroad on these bacterial toxins, it is still insufficient to identify the bio-economic factors involved in the toxin generation by bacteria from packaging to storage and consumption. Among the plant pathogenic fungi, Aspergillus spp ., Which infects all kinds of crops, has been studied in many countries, including developed countries. Many studies have reported about the biosynthesis process of toxins and the effects of toxins on their contraction. However, the research on the method of suppressing the production of mycotoxins and the removal of contaminant toxins in agricultural products is insufficient. In order to prevent the production of mycotoxins, chemical control is used to periodically spray fungicides on pre- and post-harvest agricultural products, but there is a high possibility that pesticides will remain in the agricultural products and become contaminated, resulting in pesticide toxicity in humans.

In addition, the recent foreign studies to prevent the contamination of fungal toxins such as apratoxin, there is a way to put the anti-fungal substances in cereals using genetic engineering method. Transformation of various antifungal substances, such as lyctic peptides, bacterial endo-chitinases, tobacco osmotin, etc. (Cary, JW et al., P. 16, Proceedings from the 7th Annual Aflatoxin Elim) Workshop Meeting, St. Louis, 1994; Chlan, C. et al., P. 15, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting; Li, Z. et al., P. 12, Proceedings from the 7th Annual Aflatoxin Elim Workshop Meeting). However, little is known about how such antifungal substances act as a defense against the toxicity caused by Aspergillus.

The second method is to use bioregulators to regulate the production of apratoxin. For example, the study focused on non-toxic Aspergillus, which competes with toxic bacteria. However, it is reported that the method of growing the microorganisms competing in the soil is not easy (US Pat. No. 5,942,661).

Therefore, there is a need to develop natural substances that prevent fungi from growing and producing fungal toxins in agricultural products.

The present invention has been made to solve the above-described problems, an object of the present invention is to produce a phytopathogen, especially Aspergillus spp . It is to provide an alternative to organic synthetic pesticides currently used as a control agent.

1 is a diagram showing the antiapuratoxin activity of the methanol extract extracted from the pepper against Aspergillus flavus .

2 is a diagram showing the anti-kratoxin activity of the methanol extract extracted from the pepper against Aspergillus ochraceus .

3 is a diagram showing the structure and biosynthesis process of apuratoxin B ₁ (aflatoxin B ₁ ).

In order to achieve the above object, the present invention provides a fungal toxin production inhibitor composition comprising an effective amount of pepper extract.

In the present invention, the pepper extract is preferably extracted with one or more solvents selected from hexane, methanol, ethanol, acetone, ethyl acetate, methylene chloride, chloroform and mixtures thereof.

In addition, the composition of the present invention is excellent in inhibiting fungal toxin production by using pepper extract using hexane or methanol, but is further separated from the one selected from the group consisting of piperottade Kiridine and piperongamine as active ingredients In the case of the above compound and its mixture, it has a more preferable effect.

In addition, it was confirmed that the most efficient concentration range of the extract of the present invention is 1 mg / ml to 5 mg / ml.

The compositions of the present invention have an effect on various fungal toxins and in particular have excellent effects on apuratoxins or okratoxins. Hereinafter, the present invention will be described in detail.

[Measurement of Apuratxin B ₁ Production Inhibition Activity]

First, Aspergillus flavus NRRL 2061 was used as a strain to inhibit apuratoxin B ₁ production. Keep it.

The test method was to add powder, methanol crude extract and single compound of the target plant to PDA medium, sterilize, put 10 ml into each 60 mm Petri dish, and inoculate 200 A. flavus NRRL 2061 spores into each plate medium. After 7 days of incubation at 30 ℃ is used as a sample for quantitative analysis of apuratoxin B ₁ . PDA medium without a sample was used as a control. Repeat three times for each concentration.

Quantitative analysis of apuratoxin B ₁ is performed by extracting each plate medium with 50 ml of methanol, then taking 1 ml and concentrating under nitrogen gas injection at 40 ° C. The concentrate was fixed for 10 minutes at room temperature using 200 μl of hexane and the same amount of trifluoroacetic acid and reconcentrated at 40 ° C. under nitrogen gas injection. The concentrate was dissolved in 1 ml of H ₂ O: CH ₃ CN (9: 1) mixed solvent, and 20 µl was injected into HPLC. The mobile phase of HPLC (Hewelett Packard 1050) used was H ₂ O: methanol: CH ₃ CN (60:20:20), 50 mm guard column (Ranin Instrument Co. MA, USA) and C _{18 at a} flow rate of 1.0 ml / min. Eluate using a 5 μm microsorb column (4.6 × 250 mm) (Alltech Co. IL, USA). The amount of apuratoxin B ₁ produced is measured using a fluorescence detector at 365 to 455 nm. The peak of apuratoxin B ₁ appears around 6.3.

The degree of inhibitory activity of apuratoxin B ₁ production of the sample against the apuratoxin B ₁ produced by A. flavus indicated the inhibition rate (%) relative to the control group. The inhibition rate (%) calculation formula is shown in Equation 1.

% Inhibition = [(A-B) / A] × 100 ------- (Equation 1)

A is the production amount of apuratoxin B ₁ of the control group, B is the production amount of apuratoxin B ₁ in the sampled A. flavus .

[Ochratoxin A Production Inhibition Activity Measurement]

First, strains used to inhibit the production of okratoxin A were distributed by Aspergillus ochraceus NRRL 3174 in the US Department of Agriculture, North America, USDA, Peoria, IL, USA, and lyophilized strains were maintained in agar-melt (MEA) medium. .

Experimental method was incubated for 7 days in sporulation of A. ochraceus in MEA medium, and the resulting spores were collected using 0.01% (v / v) sterilized Tween 80 solution and filtered using a sterilized cheese cloth. do. Spore concentration was counted on MEA medium and then peptone-water (0.1%, w / v) was used as diluent. Plate medium was incubated at 25 ° C. for 3 days and contained about 10 ⁶ / ml spores per plate medium.

Yeast-extract-sucrose (YES) medium was used as a basic medium for the production of okratoxin A. 50 ml of YES medium was poured into a Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes. Each sample was added to each Erlenmeyer flask by concentration, and then inoculated with a spore suspension (1 × 10 ⁶ spores / ml) and incubated at 25 ° C. for 7 days. All experiments are repeated three times.

The analysis of okratoxin A was performed by extracting the generated okratoxin A using chloroform, and each extract was concentrated under nitrogen gas injection, and the concentrate was re-dissolved with phenylacetic acid: H ₂ O (99: 1) solution. Each treated sample solution was developed on a TLC plate treated with silica gel 60 using a developing solvent of toluene: ethylacetate: formic acid (60:30:10) together with okratoxin A standard solution. Ocratoxin A shown on the TLC plate was measured using CHR-Scan densitometer (Engineering Co. Budapest, Hungary).

The results of the inhibitory effect of ochratoxin A on the production of O. latoxin A produced by A. ochraceus are expressed as% inhibition to the control group. The inhibition rate (%) calculation formula is the same as that of Equation 2.

% Inhibition = [(A-B) / A] × 100 ----- (Equation 2)

A is the production amount of okratoxin A in the control group, B is the production amount of okratoxin A in the sample A. ochraceus .

[Sequential Solvent Fraction According to Polarity]

20 g of methanol crude extract of black pepper was dissolved in 800 ml of distilled water, poured into a 2,000 ml separatory funnel, and then shaken to add the same amount of hexane. The mixture was separated into a water layer and a hexane layer, and the process was repeated. Obtain ml. Repeat 800 ml of chloroform twice in the remaining water layer to obtain 1600 ml of chloroform layer. In the remaining water layer, 800 ml of ethyl acetate was repeated twice to obtain 1,600 ml of ethyl acetate layer and water layer. As a final step, 1,600 ml of butanol and water layer were obtained in the same manner as described above. The water layer was vacuum-reduced using a lyophilizer to remove water, and the hexane layer, the chloroform layer, the ethyl acetate layer and the butanol layer were concentrated under reduced pressure to use each fraction as a sample for bioassay, separation and purification.

[Separation by Silica Gel Column Chromatography]

The hexane layer having the best insecticidal and bactericidal activity among the sequential solvent fractions according to polarity is separated by silica gel column chromatography. Glass tube column (5.5 × 70 cm, PTEE end plate attached) is selected and used according to the amount of the sample. The amount of silica gel is 50 to 60 times the amount of the sample. The main developing solvent was eluted with hexane: ethyl acetate (10: 1, 8: 1, 6: 1. 4: 1, 3: 1 and 1: 1), eluted with ethyl acetate or ethyl acetate: methanol (1: 1). do. If the sample does not dissolve in the developing solvent, dissolve it in acetone or methanol, add a small amount of silica gel, and concentrate it.

The collected sample was developed by thin layer chromatography (TLC) by mixing hexane: ethyl acetate (5: 1), ethyl acetate or ethyl acetate: methanol (1: 1) in a volume ratio, and then using TLC (SIL G / UV ₂₅₄ , Strips developed on a 0.25 mm layer with fluorescent indicator, Machereynagel Co. Germany are identified by UV lamps (UVGL-58, UV-254 / 366 nm, UVP Inc. USA), and when identified as identical spots After separation by HPLC, it is used as a sample for purification.

Hereinafter, the present invention will be described in detail through non-limiting examples.

Example 1

Isolation and Identification of Apuratoxin B ₁ , ochratoxin A Formation Inhibitors from Black Pepper ( Piper retrofractum Vahl.) Fruit

From Methanol Crude Extracts and Nucleic Acid Fractions of Black Pepper FruitsA. flavusProduces Apuratoxin B_Oneand A. ochraceusHexane fraction of methanol crude extract was apuratoxin B because it showed the inhibitory activity of ochratoxin A production._OneIt was used as a sample for the isolation and identification of, ochratoxin A production inhibitory components.

Hexane fraction (3.8 g) was separated by silica gel column chromatography. After distilling under reduced pressure for each fraction eluted using hexane, hexane: ethyl acetate (10: 1, 3: 1 and 1: 1), ethyl acetate, ethyl acetate: methanol (1: 1) and methanol as the main developing solvents, It was used as a sample of bioassay. Bioassay showed the highest inhibitory effect on the production of afuratoxin B ₁ and ochuratoxin A in the hexane: ethyl acetate (3: 1) fraction.

The hexane: ethyl acetate (3: 1) fraction, an active fraction separated by primary silica gel column chromatography, was eluted with a mixed solvent of hexane: ethyl acetate (8: 1, 6: 1, 4: 1 and 1: 1). Subsequently, the collected sample was developed by thin layer chromatography (TLC), mixed with hexane: ethyl acetate (5: 1), ethyl acetate or ethyl acetate: methanol (1: 1) in a volume ratio, and then developed on a TLC plate. They were identified by a UV lamp, and when identified as the same spot, they were combined under reduced pressure, and then subjected to apuratxin B _{1 and} ochratoxin A production inhibition activity assay. As a result, the hexane: ethyl acetate (4: 1) fraction showed strong activity, and this fraction was used as a sample for separation and purification by preparative high performance liquid chromatography.

The active fraction layer separated by secondary silica gel column chromatography (prep-HPLC) was first searched for the maximum absorption wavelength by UV spectrophotometer (λ _max = 260 nm), and preparative high performance liquid chromatography was used. Isolate and purify.

The purity of the isolated and purified compound was analyzed using high performance liquid chromatography (HPLC).

Using chromatographic and spectroscopic methods from the hexane fraction of a methanol crude extract of the pepper fruit ah furanyl toxin B ₁ and ochratoxin A production-inhibiting effect of compounds of the type excellent piperidine alkaloid piperine [compound Ⅰ, (2 E, 4 E ) - N - [5- (3,4- methylenedioxyphenyl) -2,4-pentadienoyl] piperidine], pipernonaline [ compound ⅱ, (2 E, 8 E ) - N - [9- (3,4-methylenedioxyphenyl) -2,8-nonadienoyl] piperidine], piperettine [ compound ⅲ, (2 E, 4 E , 6 E) - N - [7- (3,4- methylenedioxyphenyl) -2,4,6-heptatrienoyl] piperidine] and piperoctadecalidine [compound ⅳ (2 E, 4 E, 12 Z) - N - (2,4,12-octadecatrienoyl) piperidine] and the pyridone compound of ⅴ alkaloid piperlongumine [compound, N - (3,4,5-trimethoxycinnamoyl) -Δ ³ -pyridin-2-one] was finally isolated and purified.

TABLE 1 Antiapratoxin Activity of Black Pepper ( Piper retrofractum Vahl.) On Aspergillus flavus

Concentration (µg / ml) Inhibition of Apuratoxin B ₁ Production 250 - 500 + 1000 ++ 2000 +++ 4000 ++++ 5000 ++++

++++ in Table 1; 100 to 80%, +++; 80 to 60%, ++; 60 to 40%, +; 40 to 20%,-; It means 20 to 0%.

Table 2 Anti-apuratoxin Activity of Various Solvent Extraction Fractions of Pepper on Aspergillus flavus

Fraction Inhibition of Apuratoxin B ₁ Production Concentration (μg / ml) 2000 1000 Hexane ++++ ++++ chloroform - - Ethyl acetate - - Butanol - - water - -

In Table 2 ++++ is ah furanyl toxin B ₁ generated 100-80%, +++ is 80-60%, ++ is 60-40%, + is 40-20%, - a 20-0% Inhibited.

TABLE 3 Anti-Okratoxin Activity of Black Pepper Against Aspergillus ochraceus

Concentration (µg / ml) Inhibition of Okratoxin A Production 250 - 500 - 1000 + 2000 ++ 4000 ++++ 5000 ++++

In Table 3, ++++ shows 100-80% of okratoxin A production, +++ 80-60%, ++ 60-40%, + 40-40%,-20-20 % Deterioration is indicated.

Table 4 Anti-Okratoxin Activity of Various Solvent Extraction Fractions of Pepper on Aspergillus ochraceus

Fraction Inhibition of Okratoxin A Production Concentration (μg / ml) 2000 1000 Hexane ++++ +++ chloroform - - Ethyl acetate - - Butanol - - water - -

In Table 4, ++++ is 100-80% of okratoxin A production, 80-60% of +++, 60-40% of ++, 40-20% of +, and 20-0 of -0. % Deterioration is indicated.

TABLE 5 ¹³ C and ¹ H-NMR (CDCl ₃ ) data for piperine

number Piperine ¹³ C ¹ H One 165.2 - 2 120.0 6.36 1H d ( J = 14.6 Hz) 3 142.3 7.31 1H m 4 125.3 6.64 1H m 5 138.0 6.65 1H m One' 130.9 - 2' 105.5 6.88 1H d ( J = 1.6 Hz) 3 ' 148.1 - 4' 148.0 - 5 ' 108.3 6.67 1 H d ( J = 8.0 Hz) 6 ' 122.3 6.79 1H dd ( J = 1.6, 8.0 Hz) -OCH ₂ O- 101.2 5.86 2H s One" 43.0 3.48 2H br s 2" 25.7 1.49 2H m 3 " 24.5 1.56 2H m 4" 26.9 1.49 2H m 5 " 47.1 3.48 2H br s

Structure of piperin isolated from pepper ( Piper retrofractum Vahl.)

TABLE 6 ¹³ C and ¹ H-NMR (CDCl ₃ ) data for pipernonaline

number Pipeernonaline ¹³ C ¹ H One 165.2 - 2 120.5 5.90 1H d ( J = 15.0 Hz) 3 145.2 6.80 1H dt ( J = 15.0, 4.0 Hz) 4 32.2 6.64 1H m 5 27.9 6.65 1H m 6 28.9 7 32.6 8 128.6 6.07 1H dt ( J = 6.6, 15.4) 9 129.5 6.32 1H d ( J = 15.4) One' 132.2 - 2' 105.2 6.90 1H d ( J = 1.6 Hz) 3 ' 146.5 - 4' 147.9 - 5 ' 108.0 6.71 1H d ( J = 8.0 Hz) 6 ' 120.1 6.76 1H d ( J = 1.5 8.0 Hz) -OCH ₂ O- 100.8 5.92 2H s One" 42.9 3.48 2H br s 2" 26.5 1.49 2H m 3 " 24.6 1.56 2H m 4" 25.6 1.49 2H m 5 " 46.6 3.48 2H br s

[Formula 2] The structure of piperonarin isolated from pepper

TABLE 7 ¹³ C and ¹ H-NMR (CDCl ₃ ) data for Piperettine

number Piperettine ¹³ C ¹ H One 165.5 - 2 120.2 6.34 1 H d ( J = 14.6 Hz) 3 142.2 7.33 1H dd ( J = 11.4, 14.6 Hz) 4 130.5 6.39 1H dd ( J = 11.4, 14.0 Hz) 5 139.2 6.62 1H dt ( J = 14.0, 10.0 Hz) 6 126.8 6.64 1H dd ( J = 15.0, 10.0 Hz) 7 135.4 6.57 1H t ( J = 15.0 Hz) One' 131.6 - 2' 105.6 6.94 1H d ( J = 1.6 Hz) 3 ' 147.9 - 4' 147.9 - 5 ' 108.6 6.74 1H d ( J = 8.0 Hz) 6 ' 122.2 6.84 1H dd ( J = 1.6, 8.0 Hz) -OCH ₂ O- 101.3 5.94 2H s One" 43.4 3.48 2H br s 2" 25.7 1.48-1.60 2H m 3 " 24.8 1.61-1.70 2H m 4" 26.8 1.48-1.60 2H m 5 " 47.0 3.61 2H br s

Structure of Piperetin Isolated from Pepper

Table 8 ¹³ C and ¹ H-NMR (CDCl ₃ ) data of Piperoctade calidine

number Piperoctade calidine (Piperoctadecalidine) ¹³ C ¹ H One 165.2 - 2 118.3 6.27 1H d ( J = 14.8 Hz) 3 142.5 7.24 1H dd ( J = 14.8, 10.6 Hz) 4 128.6 6.16 1H dd ( J = 15.1, 10.6 Hz) 5 124.1 6.04 1H m 6 32.7 2.14 1H m 7-12 28.9 to 29.5 1.42-1.28 13 26.9 2.00 1H m 14 129.7 5.35 1H m 15 129.3 5.35 1H m 16 28.6 2.00 1H m 17 22.6 1.28 1H overlap 18 15.5 0.90 3H t ( J = 7.3 Hz) One' 46.5 3.61 2H br s 2' 26.4 3 ' 24.4 1.65 ~ 1.56 6H m 4' 25.4 5 ' 42.9 3.49 2H br s

[Formula 4] structure of piperotta de Kiridin isolated from pepper

Table 9 ¹³ C and ¹ H-NMR (CDCl ₃ ) data for Piperlongumine

number Piperogumin (Piperlongumine) ¹³ C ¹ H One - 2 166.7 - 3 126.5 6.02 1H dt ( J = 9.7, 1.8 Hz) 4 146.4 6.95 1H dt ( J = 9.7, 4.2 Hz) 5 25.2 2.45 2H m 6 42.1 4.04 2H t ( J = 6.3 Hz) 7 169.7 - 8 121.7 7.41 1 H d ( J = 15.6 Hz) 9 144.5 7.68 1 H d ( J = 15.6 Hz) 10 131.3 - 11 106.0 6.80 s 12 154.1 - 13 141.5 - 14 154.1 - 15 106.0 6.80 s -OCH ₃ 56.6, 61.5 3.38 s , 3.89 s

Structure of Piperogumin Isolated from Pepper

Example 2

Inhibitory Activity of Apuratoxin B ₁ Production

Apuratoxin B ₁ inhibitory activity assay of the purified and isolated hexane fraction of black pepper was carried out at the concentrations of 1,000, 500, 250, 100, 50 ㎍ / ㎖ and the results are shown in Table 10.

Apuratoxin B ₁ production inhibitory activity assay for each compound resulted in piperine, pipernonaline and piperettine inhibitory effects of 53, 56, 52% at concentrations of 1,000 μg / ml, and 34, 34, at 500 μg / ml, respectively. The production inhibitory effect was 35%, and the production inhibitory effect was 10, 25, and 9% at 250 µg / ml. However, the concentrations of 100 and 50 μg / ml did not show production inhibitory activity for the three compounds. Piperoctadecalidine and piperlongumine showed 100% production inhibition effect at the concentration of 1,000 ㎍ / mL. Piperoctadecalidine showed a 50% production inhibition effect at 100 µg / ml and a 27% production inhibition effect at 50 µg / ml. In the case of Piperlongumine, 78% of the production inhibitory effect at 100 μg / ml and 50% or more of the inhibitory effect was confirmed that the apuratoxin B ₁ production inhibitory activity was the best.

Example 3

Inhibitory Activity of Ochratoxin A Production

The inhibitory activity assay for the production of compounds isolated and purified from the hexane fraction of black pepper was carried out at the concentrations of 1,000, 500, 250, 100 ㎍ / ㎖ and the results are shown in Table 11.

The ochratoxin A inhibitory activity assay for each compound showed 50% of piperine at 50 ㎍ / mL and 50% of piperettine at 50 ㎍ / mL, respectively, and pipernonaline and piperoctadecalidine showed similar effects. It is shown in. On the other hand, piperlongumine exhibited more than 50% inhibitory effect at 250 ㎍ / ㎖, it was confirmed that the best ochratoxin A inhibitory activity.

Table 10. Anti-Apurutin Activity of Compounds Extracted from Black Pepper against Aspergillus flavus

compound Concentration (µg / ml) Production of apuratoxin B ₁ Apuratoxin production amount (㎍ / plate) Inhibition rate (%) Control 62.0 ± 0.7 Piperine 1,00050025010050 29.1 ± 0.2 ^* 40.9 ± 2.4 ^* 55.7 ± 1.9 ^* 61.9 ± 2.1 ^* 62.4 ± 2.5 ^* 53341000 Piperonarin 1,00050025010050 27.4 ± 0.8 ^* 35.3 ± 1.2 ^* 46.4 ± 0.7 ^* 55.1 ± 1.8 ^* 62.1 ± 2.1 ^* 564325110 Piperetin 1,00050025010050 29.6 ± 2.1 ^* 40.2 ± 1.2 ^* 56.7 ± 2.5 ^* 61.9 ± 2.3 ^* 61.6 ± 2.2 ^* 5235900 Piperotade Kiridine 1,00050025010050 0.0 ± 0.00 ^** 7.1 ± 1.5 ^* 15.8 ± 2.0 ^* 31.1 ± 1.7 ^* 45.2 ± 1.9 ^* 10088745027 Piperongamine 1,00050025010050 0.0 ± 0.00 ^** 3.1 ± 0.2 ^* 6.4 ± 1.2 ^* 13.9 ± 1.4 ^* 30.5 ± 2.1 ^* 10095907851

Table 11 Anti-Okratoxin Activity of Compounds Extracted from Black Pepper against Aspergillus ochraceus

compound Concentration (µg / ml) Okratoxin A production Amount of toxin (μg / plate) % Inhibition Control 23.0 ± 0.6 Piperine 1,000500250100 10.1 ± 0.7 ^* 15.9 ± 2.1 ^* 18.6 ± 0.9 ^* 22.7 ± 4.1 5032191 Pipenorraline 1,000500250100 9.8 ± 1.3 ^* 15.3 ± 2.2 ^* 17.2 ± 3.7 ^* 20.1 ± 4.0 ^* 57342513 Piperetin 1,000500250100 8.7 ± 2.1 ^* 10.2 ± 1.2 ^* 15.4 ± 2.5 ^* 19.1 ± 5.2 ^* 62563317 Piperotade Kiridine 1,000500250100 5.3 ± 0.8 ^** 10.2 ± 1.7 ^* 13.7 ± 2.5 ^* 15.9 ± 2.9 ^* 80564031 Piperongamine 1,000500250100 0.2 ± 0.01 ^** 5.6 ± 0.4 ^* 10.3 ± 0.9 ^* 12.4 ± 2.4 ^* 99765546

As can be seen in the above examples, the composition of the present invention has excellent anti-apuratoxin activity and anti-otratoxin activity, and can act as a natural physiologically active substance to replace the conventional chemical control agent.

Claims (5)

Inhibitor of fungal toxin production using piperotade Kiridine or piperongamine as an active ingredient. delete delete delete The method of claim 1, The fungal toxin is a fungal toxin production inhibitor, characterized in that the auratoxin or okra toxin.