Use of gamma-tocopherol in the prevention and treatment of plant fungal diseases and human pathogenic fungi

Abstract

The application discloses application of gamma-ambroin in prevention and treatment of plant fungal diseases and human fungal diseases. In rice blast fungus filter paper piece plate activity screening, the inhibition rate of gamma-ambroin on the rice blast fungus is 51.40%, and the activity is much higher than that of alpha-ambroin, berberine, hesperidin, eugenol and emodin methyl ether. The EC 50 50 of gamma-ambroin against the rice blast fungus is 293.55 muM, which is lower than the EC 50 50 of berberine and kasugamycin. In addition, gamma-ambroin shows inhibition activity on various plant pathogenic fungi, and can relieve pepper anthracnose disease. Meanwhile, it is found through detection that gamma-ambroin has strong inhibition activity on Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, and the MIC is 3.9-62.5 muM. In view of the high fungal inhibition activity and green safety characteristics of gamma-ambroin, the gamma-ambroin can be used in green prevention and control of plant fungal diseases and prevention of human pathogenic fungi.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product application, and particularly relates to the application of gamma-mangostin and an inhibitor taking gamma-mangostin as an effective component in preventing and treating plant fungal diseases and human pathogenic fungi. BACKGROUND

[0002] Fungi are homologous to humans in genome and belong to eukaryotes, which makes it extremely difficult to develop new antifungal drugs. Natural products are a treasure trove for new drug development and play a crucial role in the field of new drug development. With the development of isolation and activity screening technologies, more and more unique natural products with distinct mechanisms and activities are being discovered. Compared with chemically synthesized small molecules, natural products have novel structures, diversity and more unique biological activities. Therefore, it is undoubtedly an effective shortcut to find new antifungal drugs from natural products.

[0003] Garcinia mangostana L. belongs to the Garcinia genus of Clusiaceae, and the chemical composition research finds that the pericarp of G. mangostana is rich in mangostin compounds, which have antibacterial, anti-inflammatory, antioxidant and other pharmacological activities, but the application research of G. mangostana in agriculture is not much. Alpha-, beta- and gamma-mangostin are compounds isolated from the pericarp of G. mangostana, and gamma-mangostin is a hydroxylated product of alpha- and beta-mangostin. Alpha-mangostin has been patented as “Application of alpha-mangostin in preventing and treating plant diseases (CN103999859B)”, which discloses the antifungal activity of alpha-mangostin. Gamma-mangostin has been patented as “Application of mangostin in preventing and treating bacterial wilt (CN109463386A)” and “Application of plant-derived natural product gamma-mangostin in antagonizing Xanthomonas (CN115886004A)”, which discloses the application of gamma-mangostin in resisting agricultural bacteria. According to the search, there is no report on the antifungal activity of gamma-mangostin in China at present. It is found in the present application that gamma-mangostin has better antifungal activity than alpha-mangostin, and therefore can be used for preventing and treating plant diseases and human diseases caused by fungi. SUMMARY

[0004] The first object of the present application is to provide the application of gamma-mangostin in preventing and treating plant fungal diseases. The chemical structure of gamma-mangostin is shown as formula I:

[0005]

[0006] The present application provides the use of gamma-tocopherol in preventing and treating plant fungal diseases, wherein the plant fungal diseases are caused by one or more than two of the following fungi: Colletotrichum scovillei, Pseudoperonospora cubensis, Magnaporthe oryzae, Fusarium oxysporum f. sp. cubense, Botrytis cirerea, Penicillium italicum, Geotrichum citri-aurantii, Fusarium moniliforme Sheld., Bipolaris maydis (Nishik.) Shoemaker., Fusarium oxysporum f. sp. Lycopersici and Peronophythora litchii.

[0007] The prevention and treatment of plant fungal diseases is to kill and / or inhibit the growth of the fungi causing plant fungal diseases, wherein the inhibition of the growth of the fungi causing plant diseases is to inhibit the mycelium growth of the fungi and / or to inhibit the spore germination and growth of the plant pathogenic fungi.

[0008] The second object of the present application is to provide the use of gamma-tocopherol in preparing plant pathogenic fungi inhibitors, wherein the plant pathogenic fungi are one or more than two of any combination of the following fungi: Colletotrichum scovillei, Pseudoperonospora cubensis, Magnaporthe oryzae, Fusarium oxysporum f. sp. cubense, Botrytis cirerea, Penicillium italicum, Geotrichum citri-aurantii, Fusarium moniliforme Sheld., Bipolaris maydis (Nishik.) Shoemaker., Fusarium oxysporum f. sp. Lycopersici and Peronophythora litchii.

[0009] The inhibitor is a product for preventing and / or treating fungal infection of plants, including post-harvest fruits and vegetables.

[0010] A third object of the present application is to provide a plant pathogenic fungus inhibitor containing γ-amyrin as an active ingredient.

[0011] A fourth object of the present application is to provide a method for preventing and treating fungal diseases of plants, the method comprising the step of contacting γ-amyrin with the plants.

[0012] A fifth object of the present application is to provide use of γ-amyrin in the preparation of a medicine and / or a daily necessity against human pathogenic fungi.

[0013] The human pathogenic fungi include, but are not limited to, Candida albicans, Crytococcus neoformans, and Aspergillus fumigatus.

[0014] A sixth object of the present application is to provide a medicine or a daily necessity against fungal infection, containing γ-amyrin as an active ingredient.

[0015] The medicine is prepared with γ-amyrin as an active ingredient and other pharmaceutical excipients or carriers.

[0016] The dosage form of the medicine can be any one or more of granules, capsules, tablets, powders, dripping pills, sustained-release agents, or injections.

[0017] The daily necessity includes, but is not limited to, clothing disinfectant, hand sanitizer, cosmetics, shampoo, vaginal wash, or mouthwash.

[0018] The above-mentioned γ-amyrin can be obtained by chemical synthesis or extraction from Garcinia mangostana.

[0019] In order to obtain a compound with strong activity against plant pathogenic fungi, the present application screened 40 plant-derived flavonoids for activity against Pyricularia oryzae by filter paper growth rate method. It was found that the inhibition rate of γ-amyrin against Pyricularia oryzae was the highest at 51.40±2.87% at a test concentration of 5mM, while the inhibition rates of α-amyrin and hesperidin, which have been reported to have activity against plant pathogenic fungi, were 17.09±1.08% and 14.80±0.95%, respectively. The inhibition rate of β-amyrin was 6.9±3.02%, and the inhibition rates of berberine and emodin methyl ether, which are plant-derived fungicides, were 16.08±2.49% and 5.61±1.18%, respectively. This indicates that γ-amyrin has strong activity against Pyricularia oryzae. The EC50 of γ-amyrin was further determined by drug plate method.50 Value, γ-ambrettolide against Magnaporthe oryzae EC 50 is 293.55 μM, which is stronger than the biopesticide berberine (EC 50 is 955.2 μM) and kasugamycin (EC 50 is 1605.33 μM). In addition, γ-ambrettolide has broad-spectrum antibacterial activity, and has certain inhibitory activity against Colletotrichum capsici, Pseudoperonospora cubensis, Magnaporthe oryzae, Fusarium oxysporum, Botrytis cinerea, Penicillium italicum, Penicillium digitatum, Bipolaris zeicola, Bipolaris maydis, Fusarium oxysporum and Pseudoperonospora humuli. At the same time, the in vivo experiment shows that the compound has a good protective effect on pepper anthracnose.

[0020] In addition, it is found through detection that γ-ambrettolide has stronger inhibitory activity on human pathogenic fungi such as standard strains of Candida albicans (including SC5314, ATCC10231, CMCC(F)98001, BNCC186382 and BNCC337321), clinical isolates of Candida albicans (including 11D, 11E, 11F, CA632, CWQ1 and CWQ2), Cryptococcus neoformans BNCC 339771 and Aspergillus fumigatus BNCC 340016 than the positive control amphotericin B.

[0021] The above results show that γ-ambrettolide has strong broad-spectrum antibacterial activity against plant pathogenic fungi, and can be applied to prevent and treat various plant fungal diseases, meeting the current demand for creating new non-polluting plant source pesticides. At the same time, γ-ambrettolide has inhibitory activity on human pathogenic fungi, and can be used to prevent and treat human fungal diseases, meeting the current demand for the shortage of clinical antifungal drugs. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is γ-ambrettolide to relieve the disease of pepper anthracnose. DETAILED DESCRIPTION

[0023] The following examples are further illustrations of the present application, but are not intended to limit the present application.

[0024] The γ-ambrettolide involved in the present application can be obtained by chemical synthesis, extracted from mangosteen peel, or directly purchased from the market.

[0025] Example 1: Determination of the inhibitory activity of the compound on Magnaporthe oryzae by filter paper growth rate method

[0026] 1. Drug preparation

[0027] The purity of the test compounds α-amyrin, β-amyrin, γ-amyrin, berberine, tangeritin, quercetin, eugenol and physcion was greater than 95%. 20 mg of each of the above compounds was dissolved in DMSO to prepare a 50 mM test compound stock solution. The test compound stock solution was diluted with deionized water to prepare a 5 mM test compound working solution. The DMSO working solution was prepared in the same manner as the test compound working solution.

[0028] 2. Preparation of PDA solid medium

[0029] Potato 200 g was cut into small pieces, added to 800 mL of deionized water and boiled for 20 min, filtered with gauze, and 20 g of glucose and 15-20 g of agar were weighed and added to the filtrate. The volume was made up to 1 L with deionized water, and the mixture was autoclaved at high temperature and pressure.

[0030] 3. Preparation of spore suspension

[0031] Magnaporthe oryzae was inoculated on a PDA plate and incubated at 28°C for 7 days. The plate was washed with sterile water, and the spore suspension was adjusted to a concentration of 1 x 10 6 spores / mL.

[0032] 4. Experimental method

[0033] A sterilized filter paper disc with a diameter of 6 mm was placed in the middle of a PDA plate, and then 7 μL of a 5 mM test compound working solution was added dropwise. The same volume of a DMSO working solution was added dropwise as a negative control. After air-drying, 5 μL of a Magnaporthe oryzae spore suspension was added dropwise, and the plate was incubated at 28°C for 36 h. The diameter of the colonies was measured, and the inhibition rate was calculated. Inhibition rate = (colony diameter in the DMSO group - colony diameter in the compound group) / colony diameter in the DMSO group.

[0034] 5. Experimental results

[0035] Table 1. Inhibition rate of compounds against Magnaporthe oryzae

[0036]

[0037] Example 2: Determination of EC value of compounds against Magnaporthe oryzae by drug plate method 50 value

[0038] 1. Preparation of PDA drug plate

[0039] A PDA plate was prepared according to the method of Example 1, autoclaved at high temperature and pressure, and cooled to a temperature that was not hot to the touch. A test compound was added to the plate to a final concentration of 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 or 2000 μM. An equal volume of DMSO was added to the plate in which the concentration of the test compound was 0 μM. The DMSO content of all plates was not more than 1%.

[0040] 2. Experimental Methods

[0041] A sterilized filter paper disc with a diameter of 6 mm was placed in the middle of a PDA plate, and then 5 μL of a Pyricularia oryzae spore suspension (1 x 10 6 spores / mL) was added dropwise. The plate was incubated at 28°C, and when the colony diameter of the plate with a drug concentration of 0 μM was close to the diameter of the plate, the colony diameter was measured, the inhibition rate was calculated, and the inhibition curve was plotted. Inhibition rate = (colony diameter of DMSO group - colony diameter of drug group) / colony diameter of DMSO group.

[0042] 3. Experimental Results

[0043] Table 2. EC50 of compounds against Pyricularia oryzae 50 determination

[0044]

[0045] Example 3: Determination of the inhibition spectrum of compounds against agricultural fungi

[0046] The inhibition spectrum of the compounds was determined according to the filter paper disc plate rate method in Example 1. The results are shown in Table 3:

[0047] Table 3. Determination of the inhibition spectrum of compounds

[0048]

[0049] Example 4: Pepper in vivo experiment

[0050] 1. Experimental Methods

[0051] A 100 mM γ-elephantopus mother liquor (dissolved in DMSO) was prepared and diluted with deionized water (containing 0.1% Tween 20 by mass fraction). Peppers (purchased at a local wholesale market) of the same size, growth state, and without infection were washed with water and then sterilized on the surface with a 2% NaOCl solution for 2 min, and washed with sterile water three times. A 1 mm deep hole was made on the surface of the pepper using a syringe needle, and then 10 μL of the drug was added dropwise, with DMSO as a control. After 24 hours, 5 μL of a pepper anthracnose spore suspension (1 x 10 6 spores / mL) was added dropwise, and then the peppers were placed in an environment of 25°C and 85% humidity, and the lesion diameter was measured after one week.

[0052] 2. Experimental Results

[0053] As shown in Table 4, the lesion diameter of the γ-elephantopus treatment group was significantly smaller than that of the DMSO control group. Figure 1

[0054] Example 5: Determination of the inhibition activity of γ-elephantopus against human pathogenic fungi ​

[0055] 1. Strains

[0056] Candida albicans standard strain SC5314 was kindly provided by Professor Shao Jing, Anhui University of Chinese Medicine. BNCC strains were purchased from the North China Industrial Microbial Strain Engineering Technology Research Center, Henan Province. Candida albicans clinical isolates CA632, CWQ1, CWQ2, 11D, 11E, and 11F were kindly provided by Professor Chang Wenqiang, Shandong University. Other strains were laboratory preserved strains.

[0057] 2. Reagent preparation

[0058] (1) Compound stock solution and working solution preparation: 10 mg of γ-elephantopus latifolius extract powder was dissolved in 1 mL of DMSO (Macklin Company) to obtain a 10 mg / mL DMSO stock solution, which was stored at -20°C. 100 μL of the stock solution was added to 152 μL of DMSO to obtain a 10 mM γ-elephantopus latifolius working solution. The preparation method of amphotericin B (AMB) stock solution and working solution was the same as that of γ-elephantopus latifolius.

[0059] (2) RPMI-1640 medium: 10.4 g of RPMI-1640 powder and 34.53 g of MOPS powder were weighed and added to 1 L of sterilized deionized water. After complete dissolution, the pH was adjusted to 7.0 with NaOH. The sterilized solution was filtered with a 0.22 μm sterile filter membrane and stored at 4°C.

[0060] (3) YPD solid medium: 20 g of peptone, 20 g of glucose, 10 g of yeast extract, and 20 g of agar powder were weighed and added to 1 L of deionized water, which was autoclaved at high temperature and high pressure.

[0061] (4) YPD liquid medium: 20 g of peptone, 20 g of glucose, and 10 g of yeast extract were weighed and added to 1 L of deionized water, which was autoclaved at high temperature and high pressure.

[0062] 3. Experimental methods

[0063] (1) Candida albicans and Cryptococcus neoformans test method:

[0064] Candida albicans and Cryptococcus neoformans were inoculated on YPD solid medium and cultured at 30°C for 2 d. Colonies were picked and inoculated in YPD liquid medium and cultured at 30°C, 200 rpm, to the late exponential growth phase. 10 μL of the bacterial solution was inoculated again in 1 mL of YPD liquid medium and cultured at 30°C, 200 rpm, to the exponential growth phase. The cell counting plate was counted, and the bacterial solution concentration was adjusted to 1 × 10 4CFU / mL. 195 μL of the bacterial solution and 5 μL of the test compound working solution were added to the first row of the 96-well cell culture plate, and 100 μL of the bacterial solution was added to the rest of the wells. 100 μL of the liquid in the first row of the wells was transferred to the second row of the wells, and the dilution was performed in a two-fold gradient. The 96-well cell culture plate was placed in a 37°C constant temperature incubator, and the minimum inhibitory concentration (MIC) value was observed after 24 h for Candida albicans and 48 h for Cryptococcus neoformans. The growth of the bacteria in each well was observed by naked eye, and the concentration corresponding to the non-growing colony was the minimum inhibitory concentration.

[0065] (2) Aspergillus fumigatus test method:

[0066] Aspergillus fumigatus was inoculated into PDA culture medium and cultured at 30°C for 5 days. The plate was washed with PRMI-1640 medium to obtain a spore suspension, which was counted using a hemocytometer. The concentration of the spore suspension was adjusted to 1×10 5 The inhibitory activity of the compound on Aspergillus fumigatus was determined in the same way as for Candida albicans.

[0067] 4. Experimental results

[0068] Table 4. Inhibitory activity of the compound on human pathogenic fungi

[0069]

[0070] As shown in Table 4, γ-mangostin (γ-MG) has strong bactericidal activity on Cryptococcus neoformans, Aspergillus fumigatus, Malassezia and Candida albicans, and the MIC value is 3.9-62.5 μM, and the activity is stronger than that of AMB.

[0071] The above is only a preferred embodiment of the present application, and it should be noted that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined in the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. Use of γ-tocopherol in preventing and treating plant fungal diseases, wherein the γ-tocopherol has the structure as shown in Formula I: Formula I; The plant fungal diseases are caused by one or more than two of the following pathogenic fungi: Colletotrichum scovillei, Magnaporthe oryzae, Botrytis cirerea, Fusarium moniliforme Sheld., Bipolaris maydis (Nishik.) Shoemaker., Fusarium oxysporum f. sp. Lycopersici and Peronophythora litchii.

2. Use of γ-tocopherol in preparing plant pathogenic fungi inhibitor, wherein the γ-tocopherol has the structure as shown in Formula I: Formula I; The plant pathogenic fungi are one or more than two of the following: Colletotrichum scovillei, Magnaporthe oryzae, Botrytis cirerea, Fusarium moniliforme Sheld., Bipolaris maydis (Nishik.) Shoemaker., Fusarium oxysporum f. sp. Lycopersici and Peronophythora litchii.

3. A method of controlling fungal diseases of plants, characterized by, The method comprises the step of contacting γ-tocopherol with a plant, wherein the plant fungal diseases are caused by one or more than two of the following pathogenic fungi: Colletotrichum scovillei, Magnaporthe oryzae, Botrytis cirerea, Fusarium moniliforme Sheld., Bipolaris maydis (Nishik.) Shoemaker., Fusarium oxysporum f. sp. Lycopersici and Peronophythora litchii.

4. Use of γ-tocopherol in preparing medicine and / or daily necessities for resisting human pathogenic fungi, wherein the γ-tocopherol has the structure as shown in Formula I: Formula I; The human pathogenic fungi are one or more than two arbitrary combinations of the following: Crytococcus neoformans, Aspergillus fumigatus and Candida albicans.

5. Use according to claim 4, characterized in that, The dosage form of the medicine is granules, capsules, tablets, powder, drop pills, sustained-release preparations or injections; the daily necessities are laundry disinfectant, hand sanitizer, cosmetics, shampoo, vaginal wash or mouthwash.

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