A method for determining the antibacterial activity of sesame compounds from camphor trees and its application

Abstract

The application discloses a method for determining antibacterial activity of sesamin components of laurus nobilis and application thereof, and belongs to the technical field of antibacterial activity determination. The application carries out antibacterial research on laurus nobilis separation and purification products (sesamin and 9-hydroxyl sesamin), finds that 9-hydroxyl sesamin and sesamin have good inhibitory activity on mycosphaerella arachidicola, and provides a basic basis for studying the antibacterial activity of laurus nobilis.

Description

Technical Field

[0001] The invention relates to a method for determining the antibacterial activity of sesame compounds in camphor trees and an application thereof, belonging to the technical field of antibacterial activity determination. Background Art

[0002] Cinnamomum camphora, also known as multi-stemmed camphor, is a large evergreen tree in the Lauraceae family and genus Cinnamomum, named for its multiple stems. It is widely distributed in southern China and is a Class II protected wild plant. Cinnamomum camphora contains chemical components called sesamin, but the antibacterial activity of sesamin compounds has been little studied. Therefore, this study isolated and purified two compounds, 9-hydroxysesamin and sesamin, from Cinnamomum camphora for antibacterial research, providing a foundation for studying the antibacterial activity of Cinnamomum camphora. Summary of the Invention

[0003] The present invention provides a method for determining the antibacterial activity of sesame compounds in camphora, which comprises the following steps:

[0004] Step 1: Isolating and purifying two compounds from Cinnamomum camphora, namely, 9-hydroxysesamin and sesamin, and using a mycelial growth rate inhibition method to determine the antibacterial activity of 9-hydroxysesamin and sesamin against four pathogens;

[0005] Step 2: using a mycelial growth rate inhibition method to determine the toxicity of sesamin and 9-hydroxysesamin to Gliocladium nematodes;

[0006] Step 3, determination of the dry weight of mycelium by sesamin and 9-hydroxysesamin;

[0007] Step 4: Determination of the malondialdehyde content in bacterial cell membranes by using sesamin and 9-hydroxysesamin;

[0008] Step 5, determination of the relative conductivity of pathogen cell membranes by sesamin and 9-hydroxysesamin;

[0009] Step 6: Determination of the effects of sesamin and 9-hydroxysesamin on the reducing sugar content of pathogens;

[0010] Step 7: Determination of the effects of sesamin and 9-hydroxysesamin on the activity of pathogen protective enzymes.

[0011] Preferably, in step 1, the method for determining the antibacterial activity of the two compounds of 9-hydroxysesamin and sesamin against four pathogens is as follows: first, accurately weigh 3 mg of 9-hydroxysesamin and sesamin, add 2 mL of solvent (methanol: ethyl acetate = 10:1) to dissolve, and then add the prepared solution to 38 mL of liquid PDA medium to prepare a drug-containing medium with a final concentration of 75 mg / L. The blank control group is a solution with the same dissolution conditions. Use a punch with a pore diameter of 0.5 cm to punch out fungal cakes with consistent mycelial growth (apple black rot skin fungus, watermelon oxysporum Fusarium, aphaniderma, and Alternaria gloeosporium), inoculate them into the corresponding culture medium, set up 3 replicates for each treatment group, and place the inoculated bacteria in a constant temperature biochemical incubator for 6 days (temperature is 28°C).

[0012] Preferably, in step 2, the method for determining the toxicity of the sesamin and the 9-hydroxysesamin to the fungus Gliocladium nematophila is as follows: the two compounds are dissolved in methanol: ethyl acetate (10:1), and then different concentration gradients are set to prepare concentrations of 25, 50, 100, 200, and 400 μg mL －1 The drug-containing PDA medium was inoculated and cultured. The positive control group was the fungicide eugenol. The antibacterial activity of the two compounds against Gliocladium nematodes was evaluated. The logarithm of the drug concentration was used as the independent variable (x), and the inhibition rate corresponding to the concentration was used as the dependent variable (y). The toxicity regression equation, effective median concentration (EC50) value, and correlation coefficient were calculated.

[0013] Preferably, in step 3, the method for determining the dry weight of mycelia by using sesamin and 9-hydroxysesamin is as follows: sesamin and 9-hydroxysesamin are respectively formulated into drug-containing PD culture solutions with final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL. Take a strain with consistent growth, use a hole punch to take five cakes of Gliocladium nematophila pathogens and insert them into the culture medium, and the blank control is a solution with the same dissolution conditions. Place in an shaking incubator (28°C, 110r / min) and culture for 6 days, collect, clean, dry the mycelia and weigh them, and repeat three times for each treatment group.

[0014] Preferably, in step 4, the method for determining the malondialdehyde content of pathogen cell membranes of sesamin and 9-hydroxysesamin is as follows: 5 cultured bacterial cakes of Gliocladium schrenkiana pathogens are respectively taken, inoculated into PD culture medium, cultured in an oscillating incubator (28°C, 110r / min) for 6 days, filtered, and prepared to a concentration of 100μg·mL －1, take 25mL of the drug solution and add 1g of pathogenic fungus hyphae. Collect the hyphae after treating for 1, 6, 12, 18, and 24 hours at room temperature. Take 0.4g of hyphae collected at different times, add 2mL of Tris-HCl buffer to grind, centrifuge for 10 minutes, and take the supernatant for later use. Determine the malondialdehyde content of the pathogen by the thiobarbituric acid method. Add 0.5% TBA solution to 2mL of the above-mentioned mycelium extract for reaction, boil in water bath for 10 minutes, cool and then centrifuge, measure the optical density values ​​at 450, 532, and 600nm respectively, and calculate the malondialdehyde content.

[0015] Preferably, in step 5, the method for determining the relative conductivity of the pathogen cell membrane by the sesamin and the 9-hydroxysesamin is as follows: 5 cultured bacterial cakes are respectively taken, and are inoculated into PD culture medium, and cultured in an oscillating incubator (28° C., 110 r / min) for 6 days, the mycelium is washed with sterile water, and the culture medium and water on the surface of the mycelium are drained by filtration with a Buchner funnel, and then the sesamin and 9-hydroxysesamin are diluted to a concentration of 100 μg·mL －1 25 mL of each solution was added to 1 g of Gliocladium nematode mycelium. A blank control group consisted of distilled water. The samples were treated at room temperature for 0.5, 1, 1.5, 2, and 2.5 hours, and the conductivity was measured using a conductivity meter. After 2.5 hours, the mycelium was boiled and cooled, and the final conductivity was measured. Each group was repeated three times.

[0016] Preferably, in step six, the method for determining the effect of the sesamin and the 9-hydroxysesamin on the reducing sugar content of the pathogen is: measuring by the DNS method (3,5-dinitrosalicylic acid method), taking 0.1 mL of the supernatant of the mycelial extract into a clean test tube, adding 2 mL of 3,5-dinitrosalicylic acid, mixing evenly and keeping boiling in a boiling water bath for 5 minutes, taking out and rapidly cooling, adding 1 mL of Tris-HCl buffer, and measuring the absorbance value at a wavelength of 540 nm.

[0017] Preferably, in step seven, the method for determining the effects of sesamin and 9-hydroxysesamin on the activity of pathogen protection enzymes is:

[0018] (1) The effects of sesamin and 9-hydroxysesamin on catalase (CAT) activity were determined by ultraviolet spectrophotometry. 1 mL of mycelial extracts from different treatments was added with 5% titanium sulfate and concentrated ammonia solution, respectively, for reaction. After the reaction, the extracts were centrifuged at 4000 rpm for 8 minutes, and 5 mL of 5% sulfuric acid was added until completely dissolved. Distilled water was used as a blank control group. The experiment was repeated three times for each treatment group, and the absorbance was measured at 240 nm.

[0019] (2) The effects of sesamin and 9-hydroxysesamin on the activity of superoxide dismutase (SOD) were determined by using the nitroblue tetrazolium (NBT) method. 1 mL of mycelial extract from each treatment group was taken and 50 mmol·L －1 Buffer, pH 7.8, 13 mmol·L －1 Methionine, 100 nmol·L －1 EDTA solution, 75 μmol·L －1 NBT solution, 2 μmol·L －1 The riboflavin solution was mixed well. Distilled water was used as the blank control group, and the absorbance of each treatment group was measured at 560 nm.

[0020] (3) The peroxidase (POD) activity of sesamin and 9-hydroxysesamin was determined by taking 100 μL of the mycelial extract from each treatment group, adding 3 mL of pH 5.6 phosphate buffer, 1 mL of guaiacol solution, and 1 mL of 1% hydrogen peroxide, respectively. The mixture was mixed and reacted in a 38°C water bath, and the absorbance was measured at 470 nm.

[0021] Preferably, the 9-hydroxysesamin and sesamin determined by the method for determining the antibacterial activity of sesamin-like components of camphor are used in the preparation of antibacterial agents.

[0022] Preferably, the bibenzyl compound prepared by the preparation method is used in the preparation of drugs with antioxidant activity.

[0023] The present invention has carried out antibacterial research to the camphor tree separation and purification product (sesamin and 9-hydroxysesamin), and found that 9-hydroxysesamin and sesamin all have good inhibitory activity to the pathogenic fungus Gliocladium nematophylla, and the inhibition rate can reach 65.53% and 62.67% respectively, and the EC50 values ​​of both are respectively 47.291 μg / mL and 105.4 μg / mL. Sesamin and 9-hydroxysesamin have good antibacterial activity, and therefore further explore the influence of the two compounds on the cell membrane, reducing sugar content, and protective enzyme activity of the pathogen. The result shows that: the cell membrane malondialdehyde content increases along with the increase of the treatment time within a certain period of time, and both compounds can destroy the bacterial cell membrane, causing membrane lipid peroxidation. After treatment, the reducing sugar content in the pathogen body gradually decreases, and mycelial growth is suppressed. Furthermore, as treatment time increased, the activities of CAT, SOD, and POD all showed an inverted "V"-shaped trend (increasing first and then decreasing), consistent with the results of this experiment. This suggests that when G. nematodes is toxic to foreign compounds, it can mitigate the effects by regulating the activities of multiple enzymes. This study only examined the in vitro activity of 9-hydroxysesamin and sesamin against G. nematodes, providing a basic basis for studying the antibacterial activity of Cinnamomum camphora. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the inhibition rate of 9-hydroxysesamin and sesamin on four pathogens.

[0025] Figure 2 Schematic diagram of the effects of 9-hydroxysesamin and sesamin on the dry weight of pathogenic mycelium.

[0026] Figure 3 Schematic diagram of the effects of sesamin and 9-hydroxysesamin on the malondialdehyde content in cell membranes.

[0027] Figure 4 Schematic diagram of the effects of sesamin and 9-hydroxysesamin on the electrical conductivity of pathogens.

[0028] Figure 5 Schematic diagram of the effects of sesamin and 9-hydroxysesamin on the reducing sugar content of pathogen cell membranes.

[0029] Figure 6 Schematic diagram of the effects of sesamin and 9-hydroxysesamin on the activities of CAT, SOD, and POD enzymes in pathogens. DETAILED DESCRIPTION

[0030] The embodiments of the present invention will be described below with reference to the accompanying drawings.

[0031] Test methods

[0032] 2.1 Determination of the antibacterial activity of the two compounds against four pathogens

[0033] The antibacterial activity of 9-hydroxysesamin and sesamin was evaluated using the mycelial growth rate inhibition method. First, 3 mg of 9-hydroxysesamin and sesamin were accurately weighed and dissolved in 2 mL of solvent (methanol:ethyl acetate = 10:1). The prepared solution was then added to 38 mL of liquid PDA culture medium to prepare a drug-containing culture medium with a final concentration of 75 mg / L. The blank control group was a solution with the same dissolution conditions. A punch with a 0.5 cm aperture was used to punch out fungal cakes with consistent mycelial growth (apple black rot spore fungus, watermelon oxysporum Fusarium, aphanidermatum aphanidermatum, and Alternaria gloeosporium) and inoculated into the corresponding culture medium. Three replicates were set up for each treatment group, and the inoculated strains were placed in a constant temperature biochemical incubator for 6 days (temperature 28°C). The inhibition rate of the two compounds against the four strains was calculated as follows:

[0034]

[0035] 2.2 Toxicity determination of sesamin and 9-hydroxysesamin against Gliocladium nematophila

[0036] The mycelial growth rate inhibition method was used to determine the toxicity of sesamin and 9-hydroxysesamin against Gliocladium nematophila. The two compounds were dissolved in methanol:ethyl acetate (10:1) and then prepared into different concentration gradients of 25, 50, 100, 200, and 400 μg mL. －1 The drug-containing PDA medium was inoculated and cultured. The positive control group was the fungicide eugenol. The antibacterial activity of the two compounds against Gliocladium nematodes was evaluated. The logarithm of the drug concentration was used as the independent variable (x), and the inhibition rate corresponding to the concentration was used as the dependent variable (y). The toxicity regression equation, effective median concentration (EC50) value, and correlation coefficient were calculated.

[0037] 2.3 Determination of dry weight of mycelium by sesamin and 9-hydroxysesamin

[0038] To determine mycelial dry weight, sesamin and 9-hydroxysesamin were prepared in PD culture medium at final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively. Five pellets of G. nematodeum from strains with consistent growth were punched using a micropipette and inoculated into the culture medium. A blank control was prepared using a solution prepared under the same solubility conditions. The culture was incubated in a shaking incubator (28°C, 110 rpm) for 6 days. The mycelia were then harvested, washed, dried, and weighed. Each treatment group was replicated three times.

[0039] 2.4 Effects of Sesamin and 9-Hydroxysesamin on Bacterial Cell Membranes

[0040] 2.4.1 Determination of malondialdehyde content in pathogen cell membranes by sesamin and 9-hydroxysesamin

[0041] Propylene glycol content was determined by taking five cultured Gliocladium nematodes fungus cakes and inoculating them into PD culture medium. The cakes were cultured in an oscillating incubator (28°C, 110 rpm) for 6 days, filtered, and prepared into a drug concentration of 100 μg mL. －1 , take 25mL of the drug solution and add 1g of pathogenic fungus hyphae. Collect the hyphae after treating for 1, 6, 12, 18, and 24 hours at room temperature. Take 0.4g of hyphae collected at different times, add 2mL of Tris-HCl buffer to grind, centrifuge for 10 minutes, and take the supernatant for later use. Determine the malondialdehyde content of the pathogen by the thiobarbituric acid method. Add 0.5% TBA solution to 2mL of the above-mentioned mycelium extract for reaction, boil in water bath for 10 minutes, cool and then centrifuge, measure the optical density values ​​at 450, 532, and 600nm respectively, and calculate the malondialdehyde content.

[0042] 2.4.2 Determination of relative conductivity of pathogen cell membranes by sesamin and 9-hydroxysesamin

[0043] Myceilial relative conductivity determination, respectively, take 5 culture of the disease fungus fungus pie, respectively, into the PD culture solution, in the shaking incubator (28℃, 110r / min) for 6 days, with sterile water rinse mycelium, with Buchner funnel filtration to dry the mycelium surface culture solution and water, and then sesame and 9-hydroxy sesame dilution into a concentration of 100 μg·mL －1 , respectively, take 25 mL solution, 1 g of chain sporodochia myceliophthora fungus mycelium, the blank control group is distilled water. At room temperature, respectively, 0.5h, 1h, 1.5h, 2h, 2.5h after treatment, with conductivity instrument to measure the conductivity of the corresponding time, 2.5h after boiling cooling determination of the final conductivity

[15] , each group was repeated three times. Calculate the relative conductivity of mycelium, the formula is as follows:

[0044]

[0045] 2.5 sesame and 9-hydroxy sesame effect on the determination of the content of reducing sugar of the disease

[0046] Reducing sugar content determination, by DNS method (3,5-dinitrosalicylic acid method) was determined, take 0.1 mL mycelium extract supernatant in a clean test tube, add 2 mL 3,5-dinitrosalicylic acid, uniform mixing after boiling in boiling water bath for 5 min, take out and cool quickly after adding 1 mL Tris-HCl buffer, the absorbance value was determined at 540 nm.

[0047] 2.6 sesame and 9-hydroxy sesame effect on the determination of the protective enzyme activity of the disease

[0048] 2.6.1 two kinds of compounds on the effect of catalase (CAT) activity

[0049] Catalase (catalase, CAT) activity determination by ultraviolet spectrophotometry

[17] , take 1 mL of different treatment (same as 2.4.1) of mycelium extract, respectively, add 5% titanium sulfate and concentrated ammonia water reaction, after reaction 4000r / min centrifugation for 8 min, add 5% sulfuric acid 5 mL to completely dissolved, with distilled water as blank control group, each treatment group was repeated three times, the absorbance was determined at 240 nm.

[0050] 2.6.2 two kinds of compounds on the effect of superoxide dismutase (SOD) activity

[0051] Superoxide dismutase (superoxide dismutase, SOD) activity determination by nitrogen blue tetrazolium (NBT) method, take 1 mL of each treatment group (same as 2.4.1) of mycelium extract, respectively, add 50 mmol·L －1 buffer, pH 7.8 13 mmol·L －1Methionine, 100 nmol·L －1 EDTA solution, 75 μmol·L －1 NBT solution, 2 μmol·L －1 The riboflavin solution was mixed well. Distilled water was used as the blank control group, and the absorbance of each treatment group was measured at 560 nm.

[0052] 2.6.3 Effects of the two compounds on peroxidase (POD) activity

[0053] Peroxidase activity assay

[18] : Take 100 μL of mycelial extract from each treatment group (same as 2.4.1) and add 3 mL of pH 5.6 phosphate buffer, 1 mL of guaiacol solution, and 1 mL of 1% hydrogen peroxide. Mix and react in a 38°C water bath, and measure the absorbance at 470 nm.

[0054] 2.7 Data Processing and Analysis

[0055] SPSS (26.0) software was used to process the data, and Excel 2016 software was used to draw the graphs.

[0056] 3 Results and Analysis

[0057] 3.1 Results of antibacterial activity assays of sesamin and 9-hydroxysesamin against four pathogens

[0058] The inhibitory activity of 9-hydroxysesamin and sesamin against four pathogens was determined by the mycelial growth rate inhibition method. The results are as follows: at a concentration of 75 mg / L, 9-hydroxysesamin and sesamin had a very good inhibitory effect on the four pathogens, with the inhibition rates against Gliocladium fusobacteria reaching 65.53% and 62.67%, respectively. Figure 1 .

[0059] 3.2 Results of toxicity test of sesamin and 9-hydroxysesamin against Gliocladium schrenkiana

[0060] The toxicity of 9-hydroxysesamin and sesamin against G. alternariae was determined using a hyphal growth inhibition assay. The results show that both compounds exhibited inhibitory effects against G. alternariae. The EC50 values ​​for 9-hydroxysesamin and sesamin were 105.400 μg·mL⁻¹ and 47.291 μg·mL⁻¹, respectively, while the EC50 for eugenol was 30.137 μg·mL⁻¹. Sesamin exhibited slightly lower toxicity than the positive control, eugenol. The regression equation (y), correlation coefficient (r), and 95% confidence interval for the toxicity curves are shown in the table below.

[0061] Table 1 Toxicity test of sesamin and 9-hydroxysesamin against Gliocadium cateulatum

[0062]

[0063] 3.3 Effects of sesamin and 9-hydroxysesamin on the dry weight of pathogenic mycelium

[0064] After 6 days of treatment with sesamin, the mycelial growth of G. cholerae was significantly inhibited. As the concentration of 9-hydroxysesamin increased, its dry weight value became lower and lower. When the concentration of the compound was 400μg·mL-1, the dry weight of the mycelium was only 8.8mg. Similarly, after 6 days of treatment with 9-hydroxysesamin, the mycelial growth of G. cholerae was also significantly inhibited. Before the concentration of sesamin reached 50μg·mL-1, the inhibitory effect on the mycelium was not obvious, but when the concentration reached 50μg·mL-1, the dry weight of the mycelium decreased rapidly. When the concentration of the compound was 400μg·mL-1, the dry weight of the mycelium was only 27.5mg, which was significantly different from the control group. After comparison, it was found that sesamin has a greater ability to inhibit mycelial growth than 9-hydroxysesamin. (such as Figure 2 shown)

[0065] 3.4 Effects of Sesamin and 9-Hydroxysesamin on Bacterial Cell Membranes

[0066] 3.4.1 Effects of sesamin and 9-hydroxysesamin on malondialdehyde (MDA) content

[0067] In order to determine the effects of sesamin and 9-hydroxysesamin on the malondialdehyde (MDA) content of Gliocladium alternata, 100 μg / mL of sesamin and 9-hydroxysesamin were used for treatment at different time periods. The experimental results showed that, regardless of whether it was treated with sesamin or 9-hydroxysesamin, the malondialdehyde (MDA) content of the cell membrane of Gliocladium alternata increased with the increase of treatment time, reached a peak and then decreased, while the malondialdehyde (MDA) content of the control group remained almost the same. After 12 hours of treatment, the malondialdehyde (MDA) content of both groups reached the maximum. The malondialdehyde (MDA) content of the treated cell membrane was higher than that of the control group and the difference was significant, indicating that both compounds can destroy the cell membrane of the bacteria and cause membrane lipid peroxidation. (such as Figure 3 shown)

[0068] 3.4.2 Results of Sesamin and 9-Hydroxysesamin on the Relative Conductivity of Bacterial Cell Membranes

[0069] The effect of the conductivity instrument on the permeability of the cell membranes of the two pathogens was measured. The results showed that the relative conductivity of the hyphae of the two pathogens after different treatments with the two compounds increased with the increase of treatment time. When sesamin was treated for 2.5 hours, the relative conductivity reached 74.87%; when 9-hydroxysesamin was treated for 2.5 hours, the relative conductivity reached as high as 82.07%. Comparing the two groups of data, it can be found that 9-hydroxysesamin has a stronger damage to the cell membrane of the pathogen than sesamin. The data of the treatment group were greater than those of the control group throughout the whole process, indicating that both sesamin and 9-hydroxysesamin can destroy the permeability of the cell membrane, causing the solutes in the hyphae cells to leak out, and the conductivity continues to increase, thereby inhibiting the growth of hyphae. (such as Figure 4 shown)

[0070] 3.5 Effects of Sesamin and 9-Hydroxysesamin on Reducing Sugar Content in Pathogens

[0071] The experimental results show that after the Neisseria gondii fungus was treated with sesamin or 9-hydroxysesamin, the reducing sugar content in the first hour was basically the same as that in the control group, indicating that the reducing sugar content in the fungus did not change much and the mycelial growth had not been significantly inhibited. However, as the treatment time increased, the reducing sugar content in the fungus gradually decreased, while the reducing sugar content in the control group remained basically unchanged, indicating that mycelial growth was inhibited and sesamin was able to inhibit the fungus from absorbing the sugar content required for growth. It can be concluded that both sesamin and 9-hydroxysesamin can inhibit the fungus from absorbing the sugar substances required for growth. (As shown in Figure 2) Figure 5 shown)

[0072] 3.6 Effects of sesamin and 9-hydroxysesamin on the activities of pathogenic bacteria SOD, CAT, and POD

[0073] The experimental results showed that as the treatment time increased, the activities of SOD, CAT and POD enzymes showed an inverted "V" shape change, and the activities of SOD, CAT and POD enzymes were significantly different from those of the control group (such as Figure 6 In the first 18 h, the activities of SOD, CAT and POD gradually increased and reached a peak; from 18 to 24 h, the activities of SOD, CAT and POD slowly decreased, but were still higher than those in the control group. However, no significant changes occurred in the control group with the increase of treatment time, indicating that the toxic effects of Gliocladium schrenkiana on the fungus could be reduced by regulating the activities of SOD, CAT and POD after treatment with sesamin or 9-hydroxysesamin.

[0074] 4 Conclusion and Discussion

[0075] The antibacterial activity of sesame lignans (sesamin and 9-hydroxy-sesamin) was studied in this research. The results showed that 9-hydroxy-sesamin and sesamin had good inhibitory activity on the mycelial growth of H. theicola, and the inhibition rates were 65.53% and 62.67%, respectively. The EC50 values of 9-hydroxy-sesamin and sesamin were 47.291 μg / mL and 105.4 μg / mL, respectively. The good antibacterial activity of sesamin and 9-hydroxy-sesamin was further explored on the cell membrane, reducing sugar content and protective enzyme activity of the pathogen. The results showed that the cell membrane malondialdehyde content increased with the increase of treatment time within a certain time. Both compounds could destroy the cell membrane of the pathogen and cause membrane lipid peroxidation. The reducing sugar content in the pathogen decreased after treatment, and the mycelial growth was inhibited. With the increase of treatment time, the CAT, SOD and POD enzyme activities showed an inverted "V" type (first increased and then decreased) change trend, which was consistent with the experimental results. It was indicated that when the pathogen was poisoned by external compounds, it could reduce the toxic effect by adjusting the activity of various enzymes. This study only detected the in vitro activity of 9-hydroxy-sesamin and sesamin on H. theicola, which provided a basic basis for the study of the antibacterial activity of sesame lignans. Further research is needed to study the mechanism of action.

[0076] The above-described embodiments only express the preferred embodiments of the present application, which are described in detail and specifically. However, the present application is not limited to these embodiments. It should be noted that any improvement made by those skilled in the art without departing from the purpose of the present application falls within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method for determining the antibacterial activity of sesame compounds in camphor trees, characterized in that: The method for determining the antibacterial activity of sesame compounds in camphor trees comprises the following steps: Step 1, two compounds are isolated and purified from camphor, namely: 9-hydroxysesamin and sesamin, and the mycelium growth rate inhibition method is used to determine the antibacterial activity of the two compounds 9-hydroxysesamin and sesamin against four pathogens; the method for determining the antibacterial activity of the two compounds 9-hydroxysesamin and sesamin against the four pathogens is as follows: first, accurately weigh 3 mg of 9-hydroxysesamin and sesamin, add 2 mL of solvent, and dissolve them in a methanol: ethyl acetate = 10:1 configuration, and then add the prepared solution to 38 mL of liquid PDA culture medium to prepare a drug-containing culture medium with a final concentration of 75 mg / L, and the blank control group is a solution with the same dissolution conditions; use a puncher with a pore diameter of 0.5 cm to punch out apple black rot skin shell fungus, watermelon oxysporum Fusarium, aphaniderm fungus, and chain spore ... Step 2: Using the mycelial growth rate inhibition method, the toxicity of sesamin and 9-hydroxysesamin to Gliocladium nematodes was determined. The method for determining the toxicity of the sesamin and 9-hydroxysesamin to Gliocladium nematodes was as follows: the two compounds were dissolved in a methanol: ethyl acetate ratio of 10:1, and then different concentration gradients were set to prepare concentrations of 25, 50, 100, 200, and 400 μg mL. －1 The drug-containing PDA medium was inoculated and cultured; the positive control group was the fungicide eugenol. The antibacterial activity of the two compounds against Gliocladium nematodes was evaluated. The logarithm of the drug concentration was used as the independent variable, and the inhibition rate corresponding to the concentration was used as the dependent variable. The toxicity regression equation, effective median concentration value and correlation coefficient were calculated. Step 3, determination of the dry weight of mycelium by sesamin and 9-hydroxysesamin; Step 4: Determination of the malondialdehyde content in bacterial cell membranes by using sesamin and 9-hydroxysesamin; Step 5, determination of the relative conductivity of pathogen cell membranes by sesamin and 9-hydroxysesamin; Step 6: Determination of the effects of sesamin and 9-hydroxysesamin on the reducing sugar content of pathogens; Step 7: Determination of the effects of sesamin and 9-hydroxysesamin on the activity of pathogen-protective enzymes. The method for determining the effects of sesamin and 9-hydroxysesamin on the activity of pathogen-protective enzymes is as follows: (1) Determination of the activity of catalase by the sesamin and 9-hydroxysesamin, the method is as follows: using ultraviolet spectrophotometry, taking 1 mL of the mycelial extracts of different treatments, adding 5% titanium sulfate and concentrated ammonia water respectively for reaction, centrifuging at 4000 r / min for 8 minutes after the reaction, adding 5 mL of 5% sulfuric acid until completely dissolved, using distilled water as a blank control group, repeating each treatment group three times, and measuring the absorbance at 240 nm; (2) The activity of superoxide dismutase by sesamin and 9-hydroxysesamin was determined by using the nitroblue tetrazolium method. 1 mL of mycelial extract from each treatment group was taken and 50 mmol·L －1 Buffer, pH 7.8, 13 mmol·L －1 Methionine, 100 nmol·L －1 EDTA solution, 75 μmol·L －1 NBT solution, 2 μmol·L －1 The riboflavin solution was mixed; distilled water was used as the blank control group, and the absorbance value of each treatment group at 560 nm was measured respectively; (3) The peroxidase activity of sesamin and 9-hydroxysesamin was determined as follows: 100 μL of the mycelial extract of each treatment group was added with 3 mL of phosphate buffer with a pH of 5.6, 1 mL of guaiacol solution, and 1 mL of 1% hydrogen peroxide; the mixture was mixed and reacted in a water bath at 38°C, and the absorbance was measured at 470 nm.

2. The method for determining the antibacterial activity of sesame compounds according to claim 1, wherein: In step three, the method for determining the dry weight of mycelium by the sesamin and the 9-hydroxysesamin is as follows: sesamin and 9-hydroxysesamin are respectively prepared into drug-containing PD culture medium with final concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL; taking a strain with consistent growth, using a punch to take five cakes of Gliocladium nematophila pathogens and inoculating them into the culture medium, and the blank control is a solution with the same dissolution conditions; placing in an shaking incubator at 28°C and 110 r / min for 6 days, collecting, washing, drying and weighing the mycelium, and repeating three times for each treatment group.

3. A method for determining the antibacterial activity of sesame compounds according to claim 1, characterized in that: In step 4, the method for determining the malondialdehyde content of pathogen cell membranes by using sesamin and 9-hydroxysesamin is as follows: 5 cultured fungus cakes of Gliocladium schrenkiana were taken, and each was inoculated into PD culture medium, cultured in an oscillating incubator at 28°C and 110 rpm for 6 days, filtered, and prepared to a concentration of 100 μg mL －1 , take 25mL of the drug solution and add 1g of pathogen mycelium; collect the mycelium after treating at room temperature for 1, 6, 12, 18, and 24 hours respectively; take 0.4g of mycelium collected at different times, add 2mL of Tris-HCl buffer to grind, centrifuge for 10 minutes, and take the supernatant for later use; determine the malondialdehyde content of the pathogen by the thiobarbituric acid method; add 0.5% TBA solution to 2mL of the above supernatant to react, boil in water bath for 10 minutes, cool and then centrifuge, measure the optical density values ​​at 450, 532, and 600nm, respectively, and calculate the malondialdehyde content.

4. A method for determining the antibacterial activity of sesame compounds according to claim 1, characterized in that: In step 5, the method for determining the relative conductivity of the pathogen cell membrane by the sesamin and 9-hydroxysesamin is as follows: 5 cultured bacterial cakes are respectively taken, inoculated into PD culture medium, and cultured in an oscillating incubator at 28°C and 110 r / min for 6 days. The mycelium is washed with sterile water, and the culture medium and water on the surface of the mycelium are drained by filtration using a Buchner funnel. Then, the sesamin and 9-hydroxysesamin are diluted to a concentration of 100 μg·mL. －1 , take 25mL of solution respectively, add 1g of mycelium of Gliocladium schrenkiana, and the blank control group is distilled water; after treatment at room temperature for 0.5h, 1h, 1.5h, 2h, and 2.5h, the conductivity of the corresponding time is measured with a conductivity meter. After 2.5h, the mycelium is boiled and cooled to determine the final conductivity. Each group is repeated three times.

5. The method for determining the antibacterial activity of sesame compounds according to claim 1, wherein: In step six, the method for determining the effect of sesamin and 9-hydroxysesamin on the reducing sugar content of pathogens is as follows: the DNS method is used for determination, 0.1 mL of the supernatant of the mycelial extract is placed in a clean test tube, 2 mL of 3,5-dinitrosalicylic acid is added, the mixture is evenly mixed and kept boiling in a boiling water bath for 5 minutes, the mixture is taken out and quickly cooled, 1 mL of Tris-HCl buffer is added, and the absorbance value is measured at a wavelength of 540 nm.

6. Use of 9-hydroxysesamin and sesamin determined according to the method for determining the antibacterial activity of sesamin-like components of camphor trees according to any one of claims 1 to 5 in the preparation of an antibacterial agent.

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