Methods for preparing 2'-deoxyguanosine, guanosine and compositions thereof, and uses thereof

Abstract

The present invention discloses a method for preparing 2'-deoxyguanosine, guanosine and a composition thereof, and uses thereof, which belong to the technical field of controlling pathogenic microorganisms, specifically, the use of 2'-deoxyguanosine and guanosine in inhibiting Vibrio parahaemolyticus in marine fish farming, the use of 2'-deoxyguanosine and guanosine in anti-plant viruses, and the use of 2'-deoxyguanosine and guanosine in improving the content of 2'-deoxyguanosine, guanosine and salicylic acid in plants. The present invention provides a composition containing 2'-deoxyguanosine and guanosine, and a method for extracting 2'-deoxyguanosine and guanosine from Paecilomyces variotii SJ1 fungus. The present invention has found that 2'-deoxyguanosine and guanosine have an effect of controlling pathogenic microorganisms, in particular, a remarkable anti-plant virus effect, and can also improve the contents of 2'-deoxyguanosine, guanosine, and salicylic acid in plants.

Description

[Technical Field]

[0001] The present invention relates to the technical field of controlling pathogenic microorganisms, particularly to the preparation method and use of 2'-deoxyguanosine, guanosine and compositions thereof.

[0002] This invention claims priority to a Chinese patent application filed with the China Patent Office on May 9, 2022, bearing application number 202210496024.5 and entitled "Preparation method and use of 2'-deoxyguanosine, guanosine and compositions thereof," the entire contents of which are incorporated herein by reference and become part of the present invention. [Background technology]

[0003] Plant virus diseases, commonly referred to as "plant cancer," cause serious plant damage and result in huge economic losses in agricultural production. Currently, methods for controlling plant virus diseases mainly involve agricultural control, such as detoxifying seeds and seedlings, rational crop rotation, and selecting disease-resistant varieties, as well as chemical pesticides. Common pesticides used for control include guanidino-copper acetate, aminooligosaccharin, lentinan, and moroxydine hydrochloride. Chemical control remains the primary method for controlling plant virus diseases. However, there are currently no effective chemical pesticides capable of controlling plant virus diseases. Furthermore, with growing attention being paid to the development of virus resistance and the environmental pollution caused by chemical pesticides, the development of chemical-based antivirals has been significantly limited.

[0004] Nucleosides are basic structural elements that maintain the vital activities of living cells. They are small molecular compounds formed by the linkage of purine or pyrimidine bases with ribose via glycosidic bonds. Research into the pharmacological activity of nucleosides in conventional technology has mainly focused on human viral diseases and tumors, and there have been few reports on their effects on plants.

[0005] Prior art research on 2'-deoxyguanosine and guanosine has been primarily focused on the medical field. 2'-deoxyguanosine is used as an important raw material for synthesizing many antiviral and antitumor nucleic acid drugs, such as oligodeoxynucleotides, and guanosine is used as an intermediate for pharmaceuticals, such as nucleoside antiviral drugs, such as ribavirin and acyclovir. Summary of the Invention [Problem to be solved by the invention]

[0006] In response to the shortcomings of the prior art, the present invention provides methods for preparing and using 2'-deoxyguanosine, guanosine and compositions thereof.

[0007] 2'-deoxyguanosine is also known as 2'-deoxyguanine riboside. Guanosine is also called guanine riboside. [Means for solving the problem]

[0008] In a first aspect, the present invention provides use of a composition in controlling Vibrio parahaemolyticus (VP) in marine fish farming, wherein components of the composition include guanosine and 2'-deoxyguanosine.

[0009] In a second aspect, the present invention provides an anti-plant virus use of a substance, wherein the substance is guanosine or 2'-deoxyguanosine, or a composition comprising guanosine and 2'-deoxyguanosine.

[0010] According to some embodiments of the invention, the plant virus is Tobacco mosaic virus (TMV), potato virus X (PVX), cucumber mosaic virus (CMV), or potato virus Y (PVY).

[0011] According to some embodiments of the invention, the substance is used at a concentration of 150 ng / mL or greater.

[0012] According to still further embodiments of the present invention, the substance is used at a concentration of 150 ng / mL to 250 ng / mL.

[0013] According to some embodiments of the present invention, when a composition containing guanosine and 2'-deoxyguanosine is used for anti-plant virus purposes, the weight ratio of the 2'-deoxyguanosine to guanosine is (1 to 2): 1. According to still other embodiments of the present invention, the weight ratio of the 2'-deoxyguanosine to guanosine in the composition is 1:1.

[0014] In a third aspect, the present invention provides the use of a substance in increasing the content of 2'-deoxyguanosine, guanosine, and salicylic acid in a plant, wherein the substance is guanosine or 2'-deoxyguanosine, or a composition comprising guanosine and 2'-deoxyguanosine.

[0015] According to some embodiments of the present invention, in the composition containing guanosine and 2'-deoxyguanosine, the weight ratio of 2'-deoxyguanosine to guanosine is (1 to 2): 1. According to still other embodiments of the present invention, in the composition, the weight ratio of 2'-deoxyguanosine to guanosine is 1:1.

[0016] According to some embodiments of the present invention, the composition has a working concentration of 150 ng / mL or more. According to still other embodiments of the present invention, the composition has a working concentration of 150 ng / mL to 250 ng / mL.

[0017] In a fourth aspect, the present invention provides an anti-plant virus composition comprising, as ingredients, 2'-deoxyguanosine and guanosine, wherein the weight ratio of the 2'-deoxyguanosine to the guanosine is (1-2): 1. According to some embodiments of the present invention, the weight ratio of the 2'-deoxyguanosine to the guanosine in the composition is 1:1.

[0018] In a fifth aspect of the present invention, there is provided a method for extracting 2'-deoxyguanosine and guanosine from Paecilomyces variotii strain SJ1 cells, the method comprising: (1) preparing cells of Paecilomyces variotii strain SJ1 having deposit number CGMCC No. 10114, and pulverizing the cells for use; Step (2) is to mix the pulverized fungus cells from step (1) with a 10 to 50% ethanol solution at a mass / volume ratio of 1:(1 to 10) g / mL, sonicate the mixture, and then separate the mixture into solid and liquid. The liquid portion is then extracted with the Paecilomyces variotii strain SJ1 fungus cells. and step (3) subjecting the extract prepared in step (2) to semi-preparative reverse-phase HPLC chromatography to separate and prepare 2'-deoxyguanosine and guanosine.

[0019] According to some embodiments of the present invention, in step (1), the mycelium is crushed through a mesh of 50 to 70 mesh.

[0020] According to some embodiments of the present invention, in step (2), the volume fraction of the ethanol solution is 20 to 35%, and in step (2), the mass volume ratio of the bacterial cells to the ethanol solution is 1:(1 to 5) g / mL.

[0021] According to still other embodiments of the present invention, in step (2), the volume fraction of the ethanol solution is 28 to 32%.

[0022] According to some embodiments of the present invention, the ultrasonic intensity in step (2) is 1200-1600W.

[0023] According to some embodiments of the present invention, in step (2), the ultrasonic treatment time is 50 to 70 minutes.

[0024] In a sixth aspect, the present invention provides a method comprising inhibiting Vibrio parahaemolyticus using a composition, wherein the composition comprises guanosine and 2'-deoxyguanosine.

[0025] Vibrio parahaemolyticus is widely present in seawater and seafood and is prone to cause food poisoning, and therefore in some embodiments of the present invention, the method mainly comprises using the composition in marine fish culture or seafood to suppress Vibrio parahaemolyticus, for example, by adding the composition to the culture water for marine fish culture or to a storage environment (particularly a liquid environment) that comes into direct contact with the seafood.

[0026] According to some embodiments of the present invention, the weight ratio of 2'-deoxyguanosine to guanosine in the composition is (1-2): 1. According to still other embodiments of the present invention, the weight ratio of 2'-deoxyguanosine to guanosine in the composition is 1:1.

[0027] In a seventh aspect of the invention, the invention provides a method comprising applying a substance to a plant to inhibit a plant virus, wherein the substance is guanosine or 2'-deoxyguanosine, or a composition comprising guanosine and 2'-deoxyguanosine.

[0028] According to some embodiments of the present invention, the plant includes, but is not limited to, tobacco, potato, and cucumber, and according to some embodiments of the present invention, the plant may be a plant seedling, for example, a seedling that has at least grown leaves, and the application method may be, for example, a foliar spray or seed soak.

[0029] According to some embodiments of the present invention, the plant virus includes, but is not limited to, tobacco mosaic virus, potato virus X, cucumber mosaic virus, and potato virus Y.

[0030] According to some embodiments of the present invention, in the composition containing guanosine and 2'-deoxyguanosine, the weight ratio of 2'-deoxyguanosine to guanosine is (1 to 2): 1. According to still other embodiments of the present invention, in the composition, the weight ratio of 2'-deoxyguanosine to guanosine is 1:1.

[0031] According to some embodiments of the present invention, the composition has a working concentration of 150 ng / mL or more. For example, according to still other embodiments of the present invention, the composition has a working concentration of 150 ng / mL to 250 ng / mL.

[0032] In an eighth aspect, the present invention provides a method for increasing the content of 2'-deoxyguanosine, guanosine, and salicylic acid in a plant by applying a substance to the plant, the substance being guanosine or 2'-deoxyguanosine, or a composition containing guanosine and 2'-deoxyguanosine, for example, by foliar application or seed soaking.

[0033] According to some embodiments of the present invention, the plants include, but are not limited to, tobacco, potato, and cucumber.

[0034] According to some embodiments of the present invention, when a composition containing 2'-deoxyguanosine and guanosine is applied to a plant, the mass ratio of 2'-deoxyguanosine to guanosine is (1 to 2): 1. According to still other embodiments of the present invention, when a composition of 2'-deoxyguanosine and guanosine is applied to a plant, the mass ratio of 2'-deoxyguanosine to guanosine is 1:1.

[0035] According to some embodiments of the present invention, when a composition containing 2'-deoxyguanosine and guanosine is applied to a plant, the composition is used at a concentration of 150 ng / mL or more. According to still other embodiments of the present invention, when a composition of 2'-deoxyguanosine and guanosine is applied to a plant, the composition is used at a concentration of 150 ng / mL to 250 ng / mL. [Effects of the Invention]

[0036] Beneficial effects 1. In this invention, two substances, 2'-deoxyguanosine and guanosine, were identified from the extract of Paecilomyces variotii strain SJ1. Compared with other components in the extract, these two substances have significant antiviral activity. Furthermore, when these two substances are used in combination at a certain ratio, the antiviral effect is even more pronounced. This will facilitate the development and registration of new pesticides and realize industrial production. 2. The present invention also found that spraying an extract containing 2'-deoxyguanosine and guanosine on plants can increase the accumulation of 2'-deoxyguanosine, guanosine, and salicylic acid in the plants, which is beneficial for enhancing the plant's immune function.The present invention also provides an extraction method that effectively increases the content of 2'-deoxyguanosine and guanosine in an extract of Paecilomyces variotii strain SJ1. [Brief explanation of the drawings]

[0037] [Figure 1] This is a liquid chromatogram of Paecilomyces variotii SJ1 cell extract. In the figure, the chromatographic peak numbers correspond to Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, and Compound 10, respectively. [Figure 2] 2 is a mass spectrum of 2′-deoxyguanosine, which is compound 8 in FIG. 1. [Figure 3] 2 is a mass spectrum of guanosine, which is compound 10 in FIG. 1. [Figure 4] 1 shows the nuclear magnetic spectrum of compound 8, where a is the 1D 1H spectrum of 2'-deoxyguanosine and b is the 1D 13C spectrum of 2'-deoxyguanosine. [Figure 5] FIG. 1 shows the nuclear magnetic spectrum of compound 10, where a is the 1D 1H spectrum of guanosine and b is the 1D 13C spectrum of guanosine. [Figure 6] 1 is a structural diagram of 2'-deoxyguanosine and guanosine, where a is 2'-deoxyguanosine and b is guanosine. [Figure 7] 1 shows liquid chromatograms of 10 μg / mL 2'-deoxyguanosine and 10 μg / mL guanosine standards. [Figure 8] 1 is a liquid chromatogram of 2'-deoxyguanosine and guanosine in the bacterial cell extract of batch 3. [Figure 9] 1 is a liquid chromatogram showing the detection of salicylic acid in the control group CK in Example 7. [Figure 10] 1 is a liquid chromatogram showing the detection of salicylic acid of group A in Example 7. [Figure 11] 10 is a liquid chromatogram showing the detection of 2′-deoxyguanosine and guanosine in the control group CK in Example 7. [Figure 12] 10 is a liquid chromatogram in which 2′-deoxyguanosine and guanosine of group A in Example 7 are detected. [Figure 13] 1 shows plate diagrams of Vibrio parahaemolyticus, in which a is a TCBS medium plate without 2'-deoxyguanosine and guanosine, and b is a TCBS medium plate containing 1 mg / mL of 2'-deoxyguanosine and guanosine in a 1:1 ratio. DETAILED DESCRIPTION OF THE INVENTION

[0038] The technical solutions of the present invention will be further illustrated below with reference to examples, but the scope of protection of the present invention is not limited thereto.

[0039] Any content not described in detail in the examples is in accordance with the prior art in the relevant field.

[0040] Origin of the main ingredients All aminooligosaccharins were purchased from Shandong Binhaihan Biotechnology Co., Ltd. 2′-Deoxyguanosine and guanosine were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The deposit number of the Paecilomyces variotii strain SJ1 is CGMCC No. 10114, and the deposit information for this strain is published in patent document CN201510059660.1. The date of deposit was December 8, 2014, and it was deposited at the Center for Ordinary Microorganisms, China Microorganism Species Depositary (Address: No. 3, Hall, No. 1, Beichen West Road, Chaoyang District, Beijing). TCBS medium was purchased from Guangdong Huankai Microbiology Technology Co., Ltd. Paecilomyces variotii strain SJ1 will be hereinafter abbreviated as "Paecilomyces variotii strain SJ1." [Example]

[0041] Preparation of Paecilomyces variotii SJ1 cell extract The Paecilomyces variotii SJ1 strain was inoculated onto a plate of PDA medium and cultured at 25°C for 6 days. The agar mass on which the fungus had grown was removed with a punch and inoculated into a 250-mL Erlenmeyer flask containing 50 mL of PDA medium. The mixture was cultured at 28°C and 120 r / min on a rotary shaker for 3 days to prepare a seed solution. The seed solution was then inoculated into a 500-mL Erlenmeyer flask containing 150 mL of PDA medium at 10% of its volume and cultured at 28°C and 120 r / min on a rotary shaker for 5 days, after which the fermentation was stopped. The mycelium obtained by cultivation was washed, dried at 60°C, weighed, and crushed in a high-speed crusher. The crushed mycelium was then passed through a 60-mesh screen. The crushed mycelium powder was mixed with a 20% ethanol solution at a mass-volume ratio of 1:1 g / mL, and then subjected to 1200 W ultrasonic treatment for 60 minutes. The mixture was then vacuum filtered, and the filtrate was collected and used as an extract of Paecilomyces variotii SJ1 strain. [Example]

[0042] 1 mL of the Paecilomyces variotii SJ1 strain extract prepared in Example 1 was placed in a 1.5 mL centrifuge tube and centrifuged at 12,000 r / min for 10 minutes. The resulting supernatant was filtered through a 0.22 μm filter membrane. The filtrate was separated by gradient elution using reverse-phase semi-preparative HPLC chromatography to obtain 10 compounds. 10 compound samples were collected, and the liquid chromatograms and compound numbers are shown in Figure 1. [Example]

[0043] Antiviral experiments of 10 compounds isolated from the extract of Example 2 (1) Raising tobacco seedlings: Tobacco seedlings were raised using the usual raising method. (2) Inoculation and application method: After the tobacco plants had grown to 4 to 8 leaves, young tobacco seedlings were obtained. Tobacco plants with consistent growth conditions were selected. Each of the 10 compound samples isolated in Example 2 was sprayed on the tobacco leaves at a concentration of 200 ng / mL and a dosage of 10 mL. These treatment groups were designated treatment groups 1 to 10, with five tobacco plants per treatment group sprayed with water. Clean water was used as the control (CK). Each treatment was repeated three times, with each leaf evenly sprayed. Two hours after treatment, the three lower true leaves of the tobacco lines were inoculated by friction inoculation. 50 μL of the virus solution was taken and gently rubbed against the front of each leaf with 600-mesh quartz sand. The inoculated viruses were tobacco mosaic virus (TMV), potato virus X (PVX), cucumber mosaic virus (CMV), and potato virus Y (PVY). After inoculation, the plants were cultured for 5 days in an artificial climate chamber. (3) Calculation of virus content: Five days after inoculation, the leaves treated in step (2) were taken and placed under ultraviolet light to observe the fluorescence of the virus proteins in each drug treatment group. Leaves from the diseased lines with fluorescent labels were taken, labeled with aluminum foil, and quickly placed in liquid nitrogen. After all samples were processed and taken, they were placed in a refrigerator at -80°C for long-term storage. The virus content in the leaves of the virus-infected lines was measured using enzyme-linked immunosorbent assay. The reference for the antiviral experimental method is "Ultrahigh-activity immune inducer from endophytic fungi induces tobacco resistance to virus by SA pathway and RNA silencing" Peng C, Zhang A, Wang Q, et al. Ultrahigh-activity immune inducer from endophytic fungi induces tobacco resistance to virus by SA pathway and RNA silencing[J]. BMC Plant Biology, 2020, 20(1). (4) The specific detection results of the virus content are shown in Table 1. [Table 1] Analysis of the experimental data in Table 1 showed that after treating tobacco leaves with the 10 compounds, each treatment group had a certain degree of resistance to all four viruses, and the virus content was all lower than that of the control group. Of these, compound 8 had the lowest content of all four viruses and the mildest symptoms, at about 0.4 times that of the control group, followed by compound 10, with a virus content about 0.5 times that of the control group. The remaining compounds showed no significant differences compared to the control. Therefore, the present invention has determined that compounds 8 and 10 are the main antiviral substances in the Paecilomyces variotii SJ1 strain extract. [Example]

[0044] Identification of antiviral substances The two compounds with apparent resistance to viruses in Example 3 were detected using a liquid chromatography tandem mass spectrometer and a nuclear magnetic resonance spectrometer, respectively, and the structures of the two compounds with antiviral activity were identified by spectral data analysis. The liquid chromatography tandem mass spectrometry spectra and nuclear magnetic resonance spectra are shown in Figures 2, 3, 4, and 5, respectively. First, the extract was separated and purified by semi-preparative high-performance liquid chromatography. Trace amounts of impurities were present in the components obtained during this process. Second, when performing a full scan using a liquid chromatography tandem mass spectrometer, trace amounts of interfering ions were also present in the liquid chromatography tandem mass spectrometer system itself. Therefore, the mass spectra in Figures 2 and 3 contain trace amounts of other compounds in addition to 2'-deoxyguanosine and guanosine. In Figure 2, the two ions with high relative abundances, m / z 268.13 and 152.11, were estimated by database search to be the parent and child ions of 2'-deoxyguanosine, respectively. In Figure 3, the two ions with high relative abundances, m / z 284.13 and 152.16, were estimated by database search to be the parent and child ions of guanosine, respectively. Analysis revealed that compound 8 has the molecular formula C 10 H 13 N5O 4、The relative molecular weight is 267.24. ESI-MS m / z268.1[MH]+. 1H-NMR(400MHz,DMSO-d6)δ:7.93(1H,s,H-8),6.49(2H,s,NH2),5.69(1H,d,J=6.0Hz,H-1') ,4.39(1H,m,H-2'),4.08(1H,m,H-3'),3.86(1H,d,J=2.96Hz,H-4'),3.52~3.61(2H,m,Ha-5 ',Hb-5');13C-NMR(400MHz,DMSO-d6)δ:159.70(C-6),154.59(C-2),152.04(C-4),138.54( C-8),117.41(C-5),87.98(C-1'),84.80(C-4'),72.03(C-3'),62.50(C-2'),39.57(C-5'). Analysis result, compound 10は, molecular formula がC 10 H 13 N5O 5、 The relative molecular mass is 283.2であった. ESI-MS m / z284.1[MH]+. 1H-NMR(400MHz,DMSO-d6)δ:7.93(1H,s,H-8),6.49(2H,s,NH2),5.69(1H,d,J=6.0Hz,H-1') ,4.39(1H,m,H-2'),4.08(1H,m,H-3'),3.86(1H,d,J=2.96Hz,H-4'),3.52~3.61(2H,m,Ha-5 ',Hb-5');13C-NMR(400MHz,DMSO-d6)δ:157.21(C-6),154.15(C-2),151.77(C-4),135.98( C-8),117.18(C-5),86.81(C-1'),85.65(C-4'),74.15(C-3'),70.83(C-2'),61.86(C-5'). 2'-Deoxyguanosine and guanosine are known compounds, and their structures have been reported in relevant literature. In Figure 2, the m / z values ​​of the two ions with the highest relative abundances are 268.13 and 152.11. In Figure 3, the m / z values ​​of the two ions with the highest relative abundances are 284.13 and 152.16. Based on this, database searches suggested that these ions were the parent and child ions of 2'-deoxyguanosine and guanosine, respectively. Furthermore, the nuclear magnetic resonance data in Figures 4 and 5, including the hydrogen and carbon spectra, matched the data for 2'-deoxyguanosine and guanosine in the reference literature. Therefore, compound 8 was identified as 2'-deoxyguanosine (see Figure 6a for a structural diagram), and compound 10 was identified as guanosine (see Figure 6b for a structural diagram). The references are as follows: Jiao Sen, Mao Shujie, Liang Yaohua et al. Isolation and identification of nucleoside components in Acorus sieboldii and analysis of their conversion pathways [J]. Chinese Journal of Experimental Formulary, 2020, 26(12):9. Xu Xiaobo, Study on the Chemical Components of Leaves and Shoots of Gao-knot Bamboo (Phyllostachys prominens) [D]. Chinese Academy of Forestry Sciences, 2015. [Example]

[0045] Determination of 2'-deoxyguanosine and guanosine contents in Paecilomyces variotii SJ1 cell extracts Ten batches of Paecilomyces variotii SJ1 bacterial extract (1 mL each) were placed in 1.5 mL centrifuge tubes and centrifuged at 12,000 r / min for 10 minutes. The supernatant was filtered through a 0.22 μm filter membrane and subjected to analytical liquid-phase detection. The cultivation method for the 10 batches of Paecilomyces variotii SJ1 bacterial cells was the same as in Example 1. Preparation of standard solution: 1 mg of 2'-deoxyguanosine and guanosine standards, which had been dried to a constant weight, were weighed out and dissolved in water to a final volume of 100 mL to prepare a 10 μg / mL standard mother solution. Preparation of a calibration curve: 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the mother liquor of the standard were aspirated and adjusted to a volume of 1 mL to prepare standard working solutions. Using the peak time of the standard as a reference, the sample was integrated and the contents of 2'-deoxyguanosine and guanosine in the extract of Paecilomyces variotii SJ1 strain were calculated using formula (1).

number

[0046] Verification experiment of the antiviral effects of 2'-deoxyguanosine and guanosine The seedlings were treated according to the methods of raising, inoculating and applying the drug in Example 3. Group A was a solution of 2'-deoxyguanosine and guanosine at a concentration of 150 ng / mL and a mass ratio of 1:2, Group B was a solution of 2'-deoxyguanosine and guanosine at a concentration of 150 ng / mL and a mass ratio of 1:1, Group C was a solution of 2'-deoxyguanosine and guanosine at a concentration of 150 ng / mL and a mass ratio of 2:1, and Group D was a solution of 2'-deoxyguanosine and guanosine at a concentration of 150 ng / mL and a mass ratio of 3:1. The 2'-deoxyguanosine and guanosine solution at a concentration of 150 ng / mL and a mass ratio of 4:1 was used as group D, the 2'-deoxyguanosine and guanosine solution at a concentration of 150 ng / mL was used as group E, the 2'-deoxyguanosine solution at a concentration of 150 ng / mL was used as group F, the guanosine solution at a concentration of 150 ng / mL was used as group G, the aminooligosaccharin solution at a mass fraction of 5% was used as group H, and pure water was used as the control group CK. The resistance of 2'-deoxyguanosine and guanosine at each ratio to tobacco mosaic virus was studied, and the results are shown in Table 3. [Table 3] As can be seen from Table 3, both 2'-deoxyguanosine and guanosine have a control effect against tobacco mosaic virus, and the effect is greater than that of the control drug, a 5% mass fraction aminooligosaccharin solution. Under the same concentration conditions, the combined effect of 2'-deoxyguanosine standard and guanosine standard is superior to the anti-plant virus effect of 2'-deoxyguanosine or guanosine alone, proving that 2'-deoxyguanosine and guanosine have a certain synergistic effect. Furthermore, Table 3 shows that when the mass ratio of 2'-deoxyguanosine to guanosine is (1-2):1, the anti-plant virus effect is remarkable, and is superior to the anti-plant virus effect when the mass ratio of 2'-deoxyguanosine to guanosine is 4:1, and is superior to the anti-plant virus effect when the mass ratio of 2'-deoxyguanosine to guanosine is 1:2. In particular, the anti-plant virus effect is best when the mass ratio of 2'-deoxyguanosine to guanosine is 1:1. Furthermore, as can be seen from Table 3, under the same concentration conditions, the anti-plant virus effect of 2'-deoxyguanosine alone is superior to that of guanosine alone. However, when the two are applied in combination, the higher the ratio of 2'-deoxyguanosine in the composition, the better the effect. The experimental data for Groups C and E in Table 3 further prove that 2'-deoxyguanosine and guanosine have a certain synergistic effect. [Example]

[0047] Induction of changes in endogenous plant nucleosides and salicylic acid by Paecilomyces variotii SJ1 cell extracts Tobacco seedlings were grown according to standard seedling raising methods. After the tobacco plants had grown for four weeks, 10 plants were selected and sprayed with 150ng / mL 2'-deoxyguanosine and guanosine solutions at a 1:1 mass ratio (Group A) or pure water (Control CK). Each treatment was repeated three times. After 2 hours, leaves from Group A and Control CK plants were harvested and the contents of 2'-deoxyguanosine, guanosine, and salicylic acid were analyzed by liquid chromatography. (1) Extraction method for nucleoside substances: 0.50 g of tobacco leaves were crushed under liquid nitrogen conditions to form powder. 0.10 g of tobacco leaf powder was accurately weighed into a 10 mL centrifuge tube, 3 mL of pure water was added, and the mixture was ultrasonicated at room temperature for 30 minutes at a power of 250 W. After ultrasonication, the sample was centrifuged at 8000 r / min for 10 minutes, the supernatant was removed, half the volume of trichloromethane was added, the mixture was shaken for 30 minutes, the aqueous phase was aspirated, and the above procedure was repeated. The aqueous solutions extracted twice were combined, 15 mg of insoluble polyvinylpyrrolidone (PVPP) was added, the mixture was shaken up and down, and the mixture was centrifuged at 8000 r / min for 10 minutes. The supernatant was removed and filtered through a 0.22 μm microporous filter membrane for liquid detection. (2) Salicylic acid extraction method: 10.0 g of leaves were thoroughly crushed and placed in a 100 mL centrifuge tube. 4 mL of 5% trichloroacetic acid was added, and water was added up to 20 mL. 30 mL of ethyl ether was then added. The mixture was shaken thoroughly and infused for 12 hours. The mixture was then centrifuged at 8000 r / min for 5 minutes. The upper ethyl ether phase was removed, and the aqueous phase was extracted twice with ethyl ether. The combined ethyl ether phases were evaporated to dryness in a vacuum rotary evaporator. 1 mL of a mixture of 50% methanol and 50% acetate buffer (pH = 3.2) was added to dissolve the solution, and the solution was stored in an Eppendorf tube to prepare a free salicylic acid sample. The aqueous phase was adjusted to a final pH of 3.2 by adding 18.5% HCl, heated in an 80°C water bath for 1 hour, cooled, and then extracted three times with ethyl ether. The combined ethyl ether phases were evaporated to dryness, and then dissolved in 1 mL of a mixture of 50% methanol and 50% acetate buffer (pH = 3.2). The solution was then stored in an Eppendorf tube to prepare a bound salicylic acid sample. The bound salicylic acid sample was then filtered through a 0.22 μm microporous filter membrane and subjected to liquid detection. (3) The results are shown in Table 4. [Table 4] As can be seen from Table 4, when a 1:1 solution of 2'-deoxyguanosine and guanosine was sprayed, the contents of 2'-deoxyguanosine, guanosine, and salicylic acid in tobacco leaves increased by 20 to 35% compared to the control group, demonstrating that spraying a solution containing a certain ratio of 2'-deoxyguanosine and guanosine on plants causes the accumulation of 2'-deoxyguanosine, guanosine, and salicylic acid in the plants. Control group CK: The liquid chromatogram in which salicylic acid was detected is shown in Figure 9. Group A: The liquid chromatogram in which salicylic acid was detected is shown in Figure 10. Control group CK: The liquid chromatogram in which 2'-deoxyguanosine and guanosine were detected is shown in Figure 11. Group A: The liquid chromatogram in which 2'-deoxyguanosine and guanosine were detected is shown in Figure 12. [Example]

[0048] Factor selection and response surface optimization A response surface test was designed by selecting four factors, including material-to-liquid ratio, ultrasonic treatment time, ultrasonic power, and solvent concentration. The amount of nucleoside-related substances extracted was used as the response value, and the optimal extraction conditions were obtained and verified. The test design factors and results are shown in Table 5. [Table 5] [Table 6] Model creation and statistical analysis Based on the obtained data, multiple regression analysis was performed to obtain multiple quadratic regression equations between the corresponding variables of material-to-liquid ratio, ultrasonic treatment time, ultrasonic power, and solvent concentration and the extracted nucleoside content. Y=130.30+65.87*A+12.10*B+3.09*C+0.8250*D+5.89*A*B-2.39*A*C+0.2125*A*D-0.5375*B*C+6.51*B*D-1.61*C*D-1.06*A 2 -0.4208*B 2 -8.71*C 2 +2.99*D 2 The regression equation R2=0.9809, Adj R2=0.9630 indicates that there is a good correlation between the predicted values ​​of this equation and the actual values. Analysis of Variance Results: As can be seen from the analysis of variance results in Table 6, the F value of the model was 54.89, suggesting the model was significant. The P value was less than 0.0001, indicating that there was only a 0.01% chance that this F value was due to noise. The P value less than 0.05 suggests that the terms in this model were significant. In this experiment, A, B, BD, and C2 were significant terms, with the order of significance being A > B > C2 > BD, and the other terms were insignificant. The F value for lack of fit was 1, indicating that the lack of fit was not significant relative to the absolute error. There was a 53.54% chance that the F value for lack of fit was due to noise. The insignificant lack of fit suggested that this model was good. The F values ​​for each term indicated that A (material-to-liquid ratio) and B (ethanol concentration) had a significant effect on the extract concentration, with A's influence being much greater than B's. Using Design-Expert software, the optimal parameter combination was obtained: material-to-liquid ratio 1:3 (g / mL), volume concentration of ethanol 30%, extraction time 63.3 min, and ultrasonic power 1600 W. [Example]

[0049] Verification test of optimal extraction conditions The crushed powder of Paecilomyces variotii SJ1 strain was mixed with a certain concentration of ethanol solution in a certain mass-volume ratio, then ultrasonicated at a certain power for a certain period of time, and vacuum filtered to collect the filtrate for use. The filtrate was the Paecilomyces variotii SJ1 strain extract. The specific conditions were as follows, and the other conditions were the same as those of the extraction method in Example 1. The material-liquid ratio of group A was 1:3 (g / mL), the volume concentration of ethanol was 30%, the ultrasonic power was 1600 W, and the extraction time was 63.3 min. The material-liquid ratio of group B was 1:5 (g / mL), the volume concentration of ethanol was 30%, the ultrasonic power was 1600 W, and the extraction time was 63.3 min. The material-liquid ratio of group C was 1:3 (g / mL), the volume concentration of ethanol was 20%, the ultrasonic power was 1600 W, and the extraction time was 63.3 min. The material-liquid ratio for group D was 1:1 (g / mL), the volume concentration of ethanol was 20%, the ultrasonic power was 1600 W, and the extraction time was 63.3 min. The contents of 2'-deoxyguanosine and guanosine in the extracts were measured according to the steps in Example 5, and the results are shown in Table 7. [Table 7] As can be seen from the experimental data in Table 7, the extraction method of the present invention resulted in high contents of 2'-deoxyguanosine and guanosine in the extract. In particular, under the extraction conditions of a material-to-liquid ratio of 1:3 (g / mL) and an ethanol volume concentration of 30%, the contents of 2'-deoxyguanosine and guanosine in the fungal extract were the highest, with 2'-deoxyguanosine content of 10.93 mg / kg and guanosine content of 8.54 mg / kg. This means that the extraction method of the present invention is advantageous for the development of research into the subsequent production and utilization of 2'-deoxyguanosine and guanosine. [Example]

[0050] Experiments on the effects of 2'-deoxyguanosine and guanosine on Vibrio parahaemolyticus Vibrio parahaemolyticus (VP) is a common food poisoning pathogen found in coastal areas of China and is widely distributed in seawater and seafood. Vibrio parahaemolyticus was cultured at 37°C for 12 hours until the logarithmic phase, and then diluted with 0.01M pH 7.2 phosphate buffer to 10 6 The samples were diluted to 100 CFU / mL. They were inoculated into 1 mg / mL TCBS medium containing 2'-deoxyguanosine and guanosine at a 1:1 mass ratio, and into TCBS medium lacking 2'-deoxyguanosine and guanosine. After overnight incubation at 37°C, the growth of Vibrio parahaemolyticus on the medium was observed. As shown in Figure 13, compared to the control lacking 2'-deoxyguanosine and guanosine, the 1:1 ratio of 2'-deoxyguanosine and guanosine had a certain inhibitory effect on Vibrio parahaemolyticus.

[0051] The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make various modifications and variations to the present invention. Any amendments, equivalent replacements, improvements, etc. made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of a composition in suppressing Vibrio parahaemolyticus in marine fish farming, The components of the composition include guanosine and 2'-deoxyguanosine; The use, characterized in that the mass ratio of the 2'-deoxyguanosine to the guanosine is (1-2):1, and the concentration of the composition is 1 mg / mL or more.

2. The use according to claim 1, characterized in that the mass ratio of the 2'-deoxyguanosine to the guanosine is 1:

1.

3. The use according to claim 1, characterized in that the inhibition of Vibrio parahaemolyticus is achieved by adding the composition to culture water for marine fish farming or to a storage environment that comes into direct contact with seafood.

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