Application of perillyl alcohol in preparation of medicament for inhibiting growth of fusarium graminearum and medicament containing perillyl alcohol

Abstract

The invention discloses application of perillyl alcohol in preparation of a medicament for inhibiting growth of fusarium graminearum and the medicament containing the perillyl alcohol. The application mechanism is that perillyl alcohol destroys the mycelium cell membrane integrity of fusarium graminearum. According to the application disclosed by the invention, the growth inhibition effect of the perillyl alcohol on the fusarium graminearum which is a corn pathogenic bacterium is found for the first time, a series of experiments prove that the perillyl alcohol inhibits the growth of the fusarium graminearum by destroying the main component ergosterol of a fungal cell wall, and the inhibition effect is superior to that of a common bactericide carbendazim; and a foundation is laid for the development of pesticides for resisting pathogenic bacteria of corn.

Description

Technical Field

[0001] The invention relates to the technical field of pesticides, and in particular to application of perillyl alcohol in preparing a medicament for inhibiting the growth of Fusarium graminearum and a medicament containing perillyl alcohol. Background Art

[0002] Corn (Zea mays L.) is an important food crop and feed raw material in the world. It has become the first of the three major cereals in the world. However, due to its long growth cycle, it is extremely vulnerable to various diseases and pests. Among them, corn stalk rot has become the main disease that endangers corn production. Corn stalk rot is a global corn disease that harms the root system or stem base of corn caused by a variety of fungi and bacteria alone or in combination. There are two common types of bacterial stalk rot and fungal stalk rot. At present, 31 pathogenic fungi have been reported, and the most reported ones are Fusarium spp. or Pythium spp. It is generally believed that Fusarium or Pythium infection alone, Fusarium and Pythium combined infection can cause corn stalk rot. Among them, Fusarium graminearum is the main pathogen of corn stalk rot in Jilin and Henan in my country, while the main pathogens in Gansu, Sichuan, Hebei, Yunnan, Northeast China and other regions are Fusarium graminearum and Pseudomonas verticillasteris. In recent years, there have been increasing reports on bacteria that can cause corn stalk rot. The main ones reported in my country include Erwinia chrysanthemipv. zeae, Pseudomonas aeruginosa, Dickea zeae, Pantoea agglomerans, Pseudomonas extreme orientalis, Bacillus pumilus, etc.

[0003] Chemical pesticides are mainly used in agricultural production to control various pests, but excessive amounts of pesticides are used, and residual pesticides enter the soil in various ways, directly or indirectly causing toxic effects on different organisms. The problem of pesticide pollution is becoming more and more serious, and heavy metals or metalloids are often detected in pesticide products. Therefore, the synthesis of excellent and efficient chemical derivatives is of great significance for corn disease resistance. Summary of the invention

[0004] The main purpose of the present invention is to provide an application of a natural compound perillyl alcohol in the preparation of a medicament for inhibiting the growth of Fusarium graminearum and a medicament containing perillyl alcohol.

[0005] In order to achieve the above-mentioned object, the present invention provides an application of perillyl alcohol in preparing an agent for inhibiting the growth of Fusarium graminearum.

[0006] Furthermore, the mechanism of the above application is that perillyl alcohol destroys the integrity of the hyphal cell membrane of Fusarium graminearum.

[0007] The invention also provides a medicament for inhibiting the growth of Fusarium graminearum, which contains perillyl alcohol.

[0008] Furthermore, the concentration of perillyl alcohol in the agent is 1 to 7.5 μM.

[0009] Perillyl alcohol is one of the main components of the natural product perilla essential oil, and its chemical structure is shown in Formula I. Perillyl alcohol can inhibit tumorigenesis and reverse the formation of tumors. At present, there is no report on the application of perillyl alcohol in inhibiting fungi. The present invention has found through research that perillyl alcohol has a significant effect of inhibiting the growth of corn pathogenic fungus Fusarium graminearum and destroying the integrity of fungal hyphae cell membrane, and can be used to prevent and control fungal contamination in corn.

[0010]

[0011] The invention uses perillyl alcohol to inhibit the hyphae growth of Fusarium graminearum, and the toxic effect of perillyl alcohol on Fusarium graminearum is further verified by observing the microstructure of the hyphae under a scanning electron microscope.

[0012] Ergosterol is the main component of fungal cell walls and an important indicator for evaluating cell membrane integrity. The blockage of ergosterol synthesis will lead to significant impairment of cell membrane function and leakage of intracellular substances. Therefore, by measuring the ergosterol content, it is further verified that perillyl alcohol destroys the integrity of the cell membrane of Fusarium graminearum, inhibits the growth of hyphae, and achieves an antibacterial effect.

[0013] The cell membrane has the function of maintaining the intracellular environment. When the state of the cell membrane changes, the electrolytes in the cell may leak out, resulting in an increase in the relative conductivity of the extracellular environment. By measuring the relative conductivity of Fusarium graminearum after 8 hours of treatment with perillyl alcohol, it was further verified that the toxic effect of perillyl alcohol on Fusarium graminearum is to destroy the integrity of the cell membrane and inhibit the growth of hyphae.

[0014] The beneficial effects of the present invention are:

[0015] The beneficial effects of the present invention are embodied in:

[0016] The present invention is the first to discover the growth inhibitory effect of perillyl alcohol on the corn pathogenic fungus Fusarium graminearum. Through a series of experiments, it is proved that perillyl alcohol inhibits the growth of Fusarium graminearum by destroying ergosterol, the main component of the fungal cell wall, and the inhibitory effect is superior to the common fungicide carbendazim, laying a foundation for the development of pesticides against corn pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the in vitro antibacterial experiment of perillyl alcohol in Example 1.

[0018] Figure 2 This is a graph showing the effect of perillyl alcohol on the mycelial morphology of Fusarium graminearum in Example 2.

[0019] Figure 3 This is a graph showing the effect of different concentrations of perillyl alcohol on the ergosterol content of Fusarium graminearum in Example 3.

[0020] Figure 4 This is a graph showing the effect of perillyl alcohol on the extracellular conductivity of Fusarium graminearum in Example 4. DETAILED DESCRIPTION

[0021] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be noted that the following embodiments do not limit the protection scope of the present invention.

[0022] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or can be obtained by existing known methods; unless otherwise specified, the methods used in the examples of the present invention are methods known to those skilled in the art.

[0023] Example 1

[0024] In vitro antibacterial experiment of perillyl alcohol

[0025] 1.1 Experimental methods

[0026] Take 1cm 2 The Fusarium graminearum colonies were inoculated onto PDA medium and cultured in the dark at 28°C for 5 days. Perillyl alcohol was dissolved in methanol and added to the PDA medium to a final concentration of 1μM, 2.5μM, 5μM, and 7.5μM. Then 10mL of the mixture was placed in a culture dish with a diameter of 6cm. A Fusarium graminearum cake with a diameter of 3mm was obtained from the culture dish using a hole puncher. As described above, the cake was placed in the center of the PDA medium. And cultured in the dark in an incubator at 28°C for 5 days, with three replicates for each treatment. After the control group filled the culture dish, the diameter of the colony was measured by the cross method, and the inhibition rate of perillyl alcohol on Fusarium graminearum was calculated. Mycelium growth inhibition rate = (control colony diameter - treatment colony diameter) / (control colony diameter - 0.5) × 100%.

[0027] 1.2 Experimental Results

[0028] The growth inhibition rate of perillyl alcohol on Fusarium graminearum is shown in Table 1.

[0029] Table 1 Growth inhibition rate of perillyl alcohol on Fusarium graminearum

[0030]

[0031] like Figure 1 As shown in the figure, perillyl alcohol was added to the culture medium, and it was found that the inhibitory effect increased with the increase of the concentration of perillyl alcohol solution. The colony growth diameter can directly reflect the inhibitory effect of perillyl alcohol. After 5 days of culture, the colony growth diameters of the blank control group and the positive control group of D-limonene and carbendazim were 5.25cm, 4.60cm, and 4.00cm, respectively, while the colony growth diameters of the perillyl alcohol treatment groups with concentrations of 1μM, 2.5μM, 5μM, and 7.5μM were significantly smaller than those of the control group. It shows that with the increase of perillyl alcohol concentration, the growth diameter of the colony has significantly decreased. As shown in Table 1, when the concentration of perillyl alcohol is 5μM, its inhibition rate reaches 89.33%, which is 4.47 times the inhibition rate of 5μM D-limonene on the growth of Fusarium graminearum, and 2.68 times the inhibition rate of 5μM carbendazim on the growth of Fusarium graminearum. When the concentration of perillyl alcohol is 7μM, the growth of Fusarium graminearum is 100% inhibited.

[0032] Table 2 Inhibitory effect of perillyl alcohol on Fusarium graminearum

[0033]

[0034] As shown in Table 2, the EC50 of perillyl alcohol against Fusarium graminearum is 0.26 mg / L, while the EC50 value of the common fungicide carbendazim is 0.617 mg / L.

[0035] The above experiments all confirmed that perillyl alcohol has an inhibitory effect on the mycelial growth of Fusarium graminearum.

[0036] Example 2

[0037] Experimental study on the effect of perillyl alcohol on the hyphae morphology of Fusarium graminearum

[0038] 2.1 Experimental methods

[0039] Take an appropriate amount of perillyl alcohol solution and mix it evenly with the PDA medium after heating and dissolving, so that the final medium contains perillyl alcohol at a concentration of 1μM, 2.5μM, 5μM, and 7.5μM, and pour it into a sterile culture dish with a diameter of 6cm, wait for it to solidify, and set up a control group (CK). The culture dish was placed in a 28℃ constant temperature incubator for 5 days, and the bacterial cake was picked up with a syringe needle to a size of about 0.5cm×0.5cm, and clamped with tweezers into the electron microscope fixative (2.5% glutaraldehyde solution) for 4h, rinsed and soaked in 0.1M phosphate buffer at room temperature for 3 times, each time for 20min, and finally washed in 30%, 50%, 70%, 80%, 90%, and 100% ethanol for 20min respectively, and sent to the electron microscope platform for processing and observation.

[0040] 2.2 Experimental Results

[0041] Observe the untreated mycelium of Fusarium graminearum, such as Figure 2 (In the figure, A: blank control 3000×; B perillyl alcohol 1μM 3000×; C: perillyl alcohol 2.5μM 3000×; D perillyl alcohol 5μM 3000×) It was found that the mycelium was uniform in size, had many branches and a smooth surface, indicating that the mycelium was in good growth state. The mycelium treated with 5μM perillaldehyde had obvious sparse growth, abnormally enlarged protrusions appeared on the mycelium, and the mycelium surface became rough and slightly wrinkled; the mycelium treated with 7.5μM perillyl alcohol had its growth inhibited, and the mycelium was shriveled and severely wrinkled in many places.

[0042] Example 3

[0043] Effect of different concentrations of perillyl alcohol on ergosterol content in Fusarium graminearum

[0044] 3.1 Experimental methods

[0045] Pick fungi from PDA plate and inoculate into PDB medium, culture at 28℃ for 3d, collect hyphae by centrifugation at 6000r / min for 10 minutes, resuspend in 35mL pre-cooled 1×PBS buffer, centrifuge in refrigerated centrifuge (6000 / rpm), repeat 2 times, then ultrasonic treatment (ultrasonic power ratio 8%, crushing 4sec, interval 6sec, crushing 10min), centrifuge the crushed protein solution at 4℃, 6000r / min for 10min, collect supernatant, and obtain crude protein. Determine with ergosterol enzyme-linked immunosorbent assay kit:

[0046] (1) Sample addition: Set up standard wells, blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), and wells for the samples to be tested. Add 0 μl of sample diluent to the standard wells on the enzyme-labeled plate, add 40 μl of sample diluent to the wells for the samples to be tested, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well on the enzyme-labeled plate without touching the well wall as much as possible, and gently shake to mix.

[0047] (2) Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank well.

[0048] (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 min.

[0049] (4) Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water and set aside.

[0050] (5) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard. Repeat this process 5 times and pat dry.

[0051] (6) Color development: First add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and evenly develop the color at 37°C in the dark for 15 min.

[0052] (7) Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0053] (8) Measurement: Use the blank well as the zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0054] 3.2 Experimental Results

[0055] After Fusarium graminearum was treated with 1μM, 2.5μM, 5μM, and 7.5μM perillyl alcohol, the ergosterol content of Figure 3 As shown in the results, the ergosterol content in the treatment group was significantly lower than that in the control group (P < 0.05), and the ergosterol content in the fungus gradually decreased with the increase of perillyl alcohol concentration. Ergosterol is the main component of the fungal cell wall and an important indicator for evaluating the integrity of the cell membrane. The obstruction of ergosterol synthesis will lead to significant obstruction of cell membrane function and leakage of intracellular substances. It can be seen that perillyl alcohol inhibits the growth of Fusarium graminearum by destroying ergosterol, the main component of the fungal cell wall, and this inhibition is concentration-dependent.

[0056] Example 4

[0057] Experimental study on the effect of perillyl alcohol on the extracellular conductivity of Fusarium graminearum

[0058] 4.1 Experimental methods

[0059] Pick fungi from the PDA plate and inoculate them into PDB medium, culture at 28℃ for 3 days, collect mycelium by centrifugation at 6000r / min for 10 minutes, weigh 0.6g mycelium under sterile conditions and add it to 20mL sterile water, then add an appropriate amount of perillyl alcohol solution so that the final concentration of perillyl alcohol in sterile water is 1μM, 2.5μM, 5μM, and 7.5μM, respectively, and set up a control group (CK). Measure and record the conductivity value (i.e. the initial value) immediately after adding the mycelium, and measure it every 1h thereafter (i.e. the measured value), and measure it 8 times. Finally, boil it in boiling water for 10min, and measure the conductivity value after boiling (i.e. the measured value after boiling) after cooling to room temperature, and calculate the relative conductivity according to the formula. Repeat the experiment 3 times.

[0060] Relative conductivity (%) = (measured value - initial value) / (measured value after boiling - initial value) × 100%

[0061] 4.2 Experimental Results

[0062] The cell membrane has the function of maintaining the intracellular environment. When the state of the cell membrane changes, electrolytes in the cell may leak out, resulting in an increase in the relative conductivity of the extracellular environment. Figure 4 As shown in the figure, with the extension of perillyl alcohol treatment time, the relative conductivity gradually increased. When cultured to 8h, the relative conductivity of the control group was only 6.1439%, while the relative conductivity of the group treated with 7.5μM perillyl alcohol was 67.0418%, indicating that the perillyl alcohol treatment caused certain damage to the cell membrane, affected the cell membrane permeability, and led to an increase in conductivity. Moreover, the change in the permeability of the mycelial cell membrane of Fusarium graminearum was positively correlated with the concentration and action time of perillyl alcohol.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of perillyl alcohol in the preparation of agents for inhibiting the growth of Fusarium graminearum.

2. The use according to claim 1, characterized in that The mechanism of application is that perillyl alcohol destroys the integrity of the hyphal cell membrane of Fusarium graminearum.

3. An agent for inhibiting the growth of Fusarium graminearum, characterized in that: Contains perillyl alcohol.

4. The agent for inhibiting the growth of Fusarium graminearum according to claim 3, characterized in that: The concentration of perillyl alcohol in the drug is 1 to 7.5 μM.

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