Use of trans, trans-2,4-nonadienal and its clathrates in the preparation of a medicine for preventing and treating anthracnose of loquat

Abstract

The application discloses application of trans, trans-2,4-nonadienal and a clathrate thereof in preparation of a medicine for preventing and treating loquat anthracnose, and the trans, trans-2,4-nonadienal can prevent and treat the loquat anthracnose by inhibiting spore germination and mycelium growth of the anthracnose fungus and destroying cell structure to affect normal physiological functions of the anthracnose fungus; the trans, trans-2,4-nonadienal is made into a clathrate, so that the shortcomings of the trans, trans-2,4-nonadienal, such as easy volatilization, poor water solubility and irritating smell, can be overcome, the trans, trans-2,4-nonadienal can be continuously and slowly released, and the utilization efficiency of the trans, trans-2,4-nonadienal is improved; the obtained clathrate has high antibacterial activity on the anthracnose fungus, can reduce colony diameters, strongly inhibit mycelium growth, has good prevention and treatment effect on postharvest anthracnose of loquat fruits, can reduce lesion diameters of the fruits, significantly reduce disease index and rot rate of the fruits, and has better effect than the direct use of the trans, trans-2,4-nonadienal, and can be used for preparing the medicine for preventing and treating the loquat anthracnose, including but not limited to postharvest anthracnose of loquat fruits.

Description

Technical Field

[0001] This invention belongs to the field of loquat disease control and fruit preservation technology, and relates to the application of trans,trans-2,4-nonadienal (E24N) and its inclusion complex in the preparation of drugs for controlling loquat anthracnose. Background Technology

[0002] trans,trans-2,4-nonadienal is an aromatic volatile compound with a strong floral, fruity, and oily aroma. It is naturally present in various foods and plays an important role in their flavor. It is also a permitted flavoring agent in food, primarily used to formulate meat and poultry flavorings.

[0003] Anthracnose is a common plant fungal disease caused by the genus *Colletotrichum*, occurring worldwide. This genus of fungi has an extremely wide host range, infecting not only field crops and cash crops, leading to yield reduction, but also ornamental and medicinal plants, causing economic losses. Furthermore, some anthracnose fungi can remain dormant on the surface of fruits and vegetables before harvest, infecting them during storage and transportation, severely impacting their quality. In recent years, with the excessive use of chemical pesticides, some fungi in the genus *Colletotrichum* have developed resistance to common broad-spectrum fungicides, greatly increasing the difficulty of controlling plant anthracnose.

[0004] Loquat (Eriobotrya japonica) is an evergreen fruit tree originating from subtropical China, and its fruit is one of China's distinctive fruits. Loquat leaves can be used medicinally, possessing expectorant, antitussive, stomach-soothing, and gas-relieving properties. Currently, China's loquat cultivation area is approximately 2 million mu (about 133,333 hectares), with an annual output exceeding 900,000 tons, accounting for over 80% of global production. Loquat anthracnose primarily affects seedlings, leaves, and fruits. Infected seedlings suffer extensive leaf death and shedding; in severe cases, the entire seedling dies. Affected leaves develop circular to nearly circular spots, which later become grayish-white in the center and dark brown at the edges, measuring 3-7 mm in diameter. These spots can merge into larger patches as they expand. Infected fruits develop circular, light brown, water-soaked lesions that later become sunken. Pinkish, sticky granules, the conidia of the pathogen, grow on the affected area. Loquat anthracnose is the most serious disease that damages loquat fruit during post-harvest storage, transportation, and sales. Anthracnose fusiforme is one of the main septic pathogens causing loquat anthracnose. Summary of the Invention

[0005] One objective of this invention is to provide new applications for trans,trans-2,4-nonadienal and its inclusion complexes in the fields of loquat disease control and fruit preservation. Another objective is to provide a trans,trans-2,4-nonadienal inclusion complex that overcomes the inherent disadvantages of trans,trans-2,4-nonadienal, such as volatility, poor water solubility, and irritating odor, while achieving a continuous and slow release of trans,trans-2,4-nonadienal, thus improving its utilization efficiency.

[0006] Based on research, the present invention provides the following technical solution:

[0007] 1. Application of trans, trans-2,4-nonadienal and its inclusion complexes in the preparation of drugs for the prevention and treatment of loquat anthrax.

[0008] Furthermore, the pathogenic fungus causing loquat anthrax includes *Colletotrichum acutatum*.

[0009] Furthermore, the loquat anthracnose mentioned above refers to postharvest anthracnose of loquat fruit.

[0010] 2. A trans,trans-2,4-nonadienal inclusion complex, which is an inclusion complex obtained by encapsulating trans,trans-2,4-nonadienal in hydroxypropyl-β-cyclodextrin; wherein the molar ratio of trans,trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin is 1:2.

[0011] Furthermore, the trans, trans-2,4-nonadienal inclusion complex can be prepared by the following method:

[0012] S1. Dissolve hydroxypropyl-β-cyclodextrin in PBS buffer containing 20% ​​(mL / mL) ethanol at pH 6.8-7.2;

[0013] S2. Dissolve trans,trans-2,4-nonadienal in anhydrous ethanol, then add it to the hydroxypropyl-β-cyclodextrin solution obtained in step S1 and mix well; the molar ratio of trans,trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin is 1:2.

[0014] S3. Sonicate the mixture obtained in step S2 at 65W power for 25-35 minutes;

[0015] S4. Place the mixture after ultrasonic treatment in step S3 at 4℃ to 8℃ for 18-24 hours, filter it with a 0.2μm microporous membrane, and collect the filtrate.

[0016] S5. The filtrate obtained in step S4 is freeze-dried under vacuum at -50℃ to -60℃ for 36-48 hours to obtain the inclusion complex.

[0017] Preferably, in the preparation method, the ratio of hydroxypropyl-β-cyclodextrin to PBS buffer is 0.1 mmol: 60 mL, and the ratio of trans, trans-2,4-nonadienal to anhydrous ethanol is 0.05 mmol: 2 mL.

[0018] Preferably, in the preparation method, the ultrasonic treatment is carried out continuously for 30 minutes at a power of 65W, with a working and pausing pattern of 1 second on and 1 second off.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) This invention provides the application of trans, trans-2,4-nonadienal in the preparation of drugs for the prevention and treatment of loquat anthracnose. It affects the normal physiological function of loquat anthracnose fungus by inhibiting the germination of anthracnose spores and the growth of hyphae and destroying cell structure, thereby preventing and treating loquat anthracnose through direct pathogen inhibition.

[0021] (2) trans,trans-2,4-nonadienal is volatile and has poor stability under light and high temperature, making it difficult to preserve. Its weak hydrophilicity leads to poor biocompatibility with fruits and vegetables. Its strong aromatic odor may cause some irritation to consumers. Constructing trans,trans-2,4-nonadienal into an inclusion complex can significantly improve stability and shelf life, reduce volatility, and effectively mask the original irritating odor. It can also improve water solubility and biocompatibility with fruits and vegetables. Furthermore, it can achieve the effect of continuous and slow release of trans,trans-2,4-nonadienal, thereby improving utilization efficiency.

[0022] (3) The present invention provides a trans, trans-2,4-nonadienal inclusion complex, which is obtained by encapsulating trans, trans-2,4-nonadienal in hydroxypropyl-β-cyclodextrin. The encapsulation rate is about 90% and the loading rate is about 3.5%. The preparation method is simple and low in cost.

[0023] (4) The trans, trans-2,4-nonadienal inclusion complex of the present invention exhibits high antibacterial activity against loquat anthracnose fungus, can reduce the colony diameter, strongly inhibit mycelial growth, and has a good control effect on postharvest anthracnose of loquat fruit. It can reduce the diameter of fruit lesions, significantly reduce the disease index and rot rate of fruit, and its effect is better than the direct use of trans, trans-2,4-nonadienal. It can be used to prepare drugs for the prevention and control of loquat anthracnose, including but not limited to postharvest anthracnose of loquat fruit. Attached Figure Description

[0024] Figure 1 The effect of trans,trans-2,4-nonadienal on the growth of anthrax bacteria colonies is shown in Figure a, where a is a colony photograph and b is the colony diameter.

[0025] Figure 2 The effect of trans,trans-2,4-nonadienal on the germination of anthracnose spores is shown in Figure a, where a is a microscopic observation and b is the spore germination rate.

[0026] Figure 3 The effect of trans,trans-2,4-nonadienal on the cell membrane of Bacillus anthracis.

[0027] Figure 4 The effect of the molar ratio of trans,trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin on the encapsulation efficiency (a) and loading rate (b) of the inclusion complex.

[0028] Figure 5 Scanning electron microscope image of the trans,trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex.

[0029] Figure 6 The effect of the trans,trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex on the mycelial growth of Bacillus anthracis is shown in Figure a, where a is a colony photograph and b is the colony diameter.

[0030] Figure 7 The direct inhibitory effect of the trans,trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex on loquat anthracnose pathogen is shown in Figure a, where a is a photograph of the treated fruit and b is the diameter of the lesion.

[0031] Figure 8 The study aimed to investigate the control effect of fumigation with trans,trans-2,4-nonadienal and its inclusion complexes on postharvest anthracnose of loquat fruit. In the figure, a is a photograph of the treated fruit, b is the disease index, and c is the rot rate. Detailed Implementation

[0032] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below.

[0033] Experimental Example 1: Effect of trans,trans-2,4-nonadienal on the mycelial growth of Bacillus anthracis

[0034] Trans- and trans-2,4-nonadienal were dissolved in anhydrous ethanol and added to potato dextrose agar (PDA) medium to achieve final concentrations of 0.1, 0.2, 0.3, and 0.4 μL / mL, respectively, forming four experimental groups (0.1E24N, 0.2E24N, 0.3E24N, and 0.4E24N). An equal volume of anhydrous ethanol was added to the PDA medium as a control group. A 5mm × 5mm mycelial disc of *Anthracis humicus* with a mycelial age of 7 days was taken and inoculated onto the center of each of the five culture plates. The plates were incubated at 25°C for 7 days, and the colony diameter was photographed and recorded.

[0035] The results are as follows Figure 1 As shown. By Figure 1 As shown in Figure a, the inhibitory effect of trans,trans-2,4-nonadienal on *Anthracis caudatus* increases with increasing concentration; the culture medium containing 0.4 μL / mL trans,trans-2,4-nonadienal completely inhibited the growth of *Anthracis caudatus*. Figure 1As shown in b, after 7 days of culture, the colony diameter in the control group reached 71 mm, while the colony diameters in the 0.1E24N group, 0.2E24N group, and 0.3E24N group were 63 mm, 49 mm, and 36 mm, respectively, which were 11.26%, 30.99%, and 49.30% smaller than those in the control group. This indicates that trans, trans-2,4-nonadienal has a good inhibitory effect on the mycelial growth of *Anthracis cibarius*.

[0036] Experimental Example 2: Effect of trans,trans-2,4-nonadienal on anthracnose spore germination

[0037] Trans- and trans-2,4-nonadienal were diluted with an aqueous solution containing 20% ​​(ml / ml) ethanol and added to potato dextrose broth (PDB medium) to achieve final concentrations of 0.05, 0.1, and 0.2 μL / mL, respectively, serving as three experimental groups (0.05 μL / mL, 0.1 μL / mL, and 0.2 μL / mL). Simultaneously, an equal volume of an aqueous solution containing 20% ​​(ml / ml) ethanol was added to the PDB medium as a control group. Anthracnose spores were added to the above four PDB broths and incubated on a shaker at 25°C for 12 h. Bacterial samples were collected at 6 h, 9 h, and 12 h of incubation, and the spore germination rate was observed under a microscope and calculated.

[0038] The results are as follows Figure 2 As shown. By Figure 2 As shown in Figure a, after 12 hours of cultivation, the germination rate of *Anthracis niger* spores in all three experimental groups was significantly lower than that in the control group, indicating that trans,trans-2,4-nonadienal can inhibit the germination of *Anthracis niger* spores. Figure 2 As shown in b, the germination rate of *Anthracis niger* spores in the control group reached 85% after 9 hours of culture, while only the 0.05 μL / mL group of the three experimental groups showed germination of *Anthracis niger* spores, and the other two experimental groups did not show germination. This indicates that trans,trans-2,4-nonadienal has a good inhibitory effect on the germination of *Anthracis niger* spores.

[0039] Experimental Example 3: Effects of trans,trans-2,4-nonadienal on the cell membrane of Bacillus anthracis

[0040] Take 1.5g of *Anthracis cibarius* hyphae cultured for 7 days and suspend them in 30mL of PBS buffer containing 0, 0.1, 0.2, and 0.4μL / mL trans, trans-2,4-nonadienal, respectively. Incubate in a shaker at 25℃ and 150r / min for 6h. Collect the hyphae, stain with 500μL propidium iodide in the dark for 20min, wash three times with PBS buffer, and observe under a fluorescence upright microscope.

[0041] The results are as follows Figure 3As shown in the figure, in the control group (without trans-,trans-2,4-nonadienal), no obvious red fluorescence reaction was observed in the hyphae and spores of *Anthracis oxysporum*. However, in the three experimental groups with trans-,trans-2,4-nonadienal concentrations of 0.1, 0.2, and 0.4 μL / mL, the red fluorescence intensity of *Anthracis oxysporum* hyphae and spores showed an increasing trend with increasing trans-,trans-2,4-nonadienal concentration. This phenomenon indicates that trans-,trans-2,4-nonadienal treatment led to the destruction of the cell structure of *Anthracis oxysporum*.

[0042] Preparation Example 1: Effect of the molar ratio of trans,trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin on the encapsulation efficiency and loading rate of the inclusion complex.

[0043] Includes the following steps:

[0044] S1. Add 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, and 0.3 mmol of hydroxypropyl-β-cyclodextrin to 60 ml of PBS buffer containing 20% ​​(ml / ml) ethanol at pH 6.8-7.2, and stir at 2000 rpm for 12 min to completely dissolve the hydroxypropyl-β-cyclodextrin.

[0045] S2. Dissolve 0.05 mmol of trans,trans-2,4-nonadienal in 2 ml of anhydrous ethanol, and then slowly add it dropwise to the hydroxypropyl-β-cyclodextrin solution obtained in step S1, and mix well.

[0046] S3. The mixture obtained in step S2 is ultrasonically treated for 30 minutes at 65W power using an ultrasonic cell disruptor in a 1-second work-1-second pause mode;

[0047] S4. Place the mixture after ultrasonic treatment in step S3 at 4°C to 8°C for 24 hours, filter it with a 0.2μm microporous membrane, and collect the filtrate.

[0048] S5. The filtrate obtained in step S4 is freeze-dried under vacuum at -60℃ for 48h to obtain inclusion complexes of trans, trans-2,4-nonadienal and hydroxypropyl-β-cyclodextrin in different molar ratios.

[0049] After treatment with acetonitrile, the encapsulation efficiency and loading rate of the obtained inclusion complex were calculated by measuring its ultraviolet absorbance. The results are as follows: Figure 4As shown, the encapsulation efficiency of the inclusion complex increases with the increasing molar ratio of trans,trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin, reaching a peak of 89.19% when the molar ratio is 1:2. The loading rate decreases with increasing molar ratio, reaching 3.49% when the molar ratio is 1:2. Considering both encapsulation efficiency and loading rate, a molar ratio of trans,trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin of 1:2 is preferred for preparing the inclusion complex.

[0050] Preparation Example 2: Preparation of trans,trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex (E24N / HPβCD-IC)

[0051] Includes the following steps:

[0052] S1. Add 0.1 mmol of hydroxypropyl-β-cyclodextrin to 60 ml of PBS buffer containing 20% ​​(ml / ml) ethanol at pH 6.8-7.2, and stir at 2000 rpm for 12 min to completely dissolve the hydroxypropyl-β-cyclodextrin.

[0053] S2. Dissolve 0.05 mmol of trans,trans-2,4-nonadienal in 2 ml of anhydrous ethanol, and then slowly add it dropwise to the hydroxypropyl-β-cyclodextrin solution obtained in step S1, and mix well.

[0054] S3. The mixture obtained in step S2 is ultrasonically treated for 30 minutes at 65W power using an ultrasonic cell disruptor in a 1-second work-1-second pause mode;

[0055] S4. Place the mixture after ultrasonic treatment in step S3 at 4°C to 8°C for 24 hours, filter it with a 0.2μm microporous membrane, and collect the filtrate.

[0056] S5. The filtrate obtained in step S4 is freeze-dried under vacuum at -60℃ for 48 hours to obtain the inclusion complex.

[0057] The obtained inclusion complex was a white powdery solid. After gold plating, its morphology was observed using a scanning electron microscope. The results are as follows: Figure 5 As shown, the inclusion complex E24N / HPβCD-IC is an irregular sheet and block shape of varying sizes, exhibiting a significant morphological change compared to hydroxypropyl-β-cyclodextrin (HPβCD). Preliminary analysis suggests that the inclusion complex prepared by the freeze-drying method represents a new phase, indicating that trans-,trans-2,4-nonadienal interacts with hydroxypropyl-β-cyclodextrin molecules, altering the original morphology of hydroxypropyl-β-cyclodextrin.

[0058] Experimental Example 4: Effect of trans,trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex on the mycelial growth of Bacillus anthracis.

[0059] The inclusion complex prepared in Preparation Example 2 was added to PDA medium to achieve final concentrations of 1, 2.5, and 5 mg / mL, forming three experimental groups. A control group (PDA medium without the inclusion complex) was also established. 1×10⁶ cells / mL were inoculated at the center of each of the four culture plates. 6 spores·mL -1 10 μL of anthrax spore suspension was placed in a constant temperature incubator at 25℃ for 7 days, and the colony diameter was photographed and recorded.

[0060] The results are as follows Figure 6 As shown. By Figure 6 As shown in section a, the inhibitory effect of the inclusion complex on *Anthracis humicis* increases with increasing concentration; the culture medium containing 5 mg / mL of the inclusion complex completely inhibited the growth of *Anthracis humicis*. Figure 6 b shows that after 7 days of culture, the colony diameter in the control group reached 72 mm, while the colony diameters in the 1 mg / mL and 2.5 mg / mL inclusion complex groups were 63 mm and 58 mm, respectively, which were 12.50% and 19.44% smaller than those in the control group. This indicates that the trans, trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex can effectively inhibit the mycelial growth of Bacillus oxysporum.

[0061] Experimental Example 5: Direct inhibitory effect of trans,trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex on loquat anthracnose pathogen.

[0062] Dissolve 500 mg of the inclusion complex prepared in Example 2 in 1 mL of deionized water to prepare an aqueous solution of the inclusion complex. Select loquat fruits of uniform size and maturity, soak them in 0.1% sodium hypochlorite solution for 10 min, rinse twice with water, and then air dry. Make one hole at the equator on each fruit, and inoculate using the same hole method, inoculating each wound with 10 sachets. 5 spores·mL -1 Five μL of *Anthracnose spores* solution was added to each wound 4 hours later, followed by 20 μL of the above inclusion complex aqueous solution as the treatment group (E24N / HPβCD-IC). A control group (control) was also included, with 20 μL of deionized water added. Loquat fruits from both groups were placed in sealed fruit boxes and stored at 25°C for 7 days. The diameter of the lesions was then measured.

[0063] The results are as follows Figure 7 As shown, in loquat fruits infected with *Anthracis cibarius*, the average diameter of lesions in the control group was 22 mm, while the average diameter of lesions in the treatment group with the inclusion complex E24N / HPβCD-IC was 13 mm, a reduction of 34.45%. This indicates that the trans, trans-2,4-nonadienal / hydroxypropyl-β-cyclodextrin inclusion complex exhibits significant biological activity in inhibiting anthracis infection.

[0064] Experimental Example 6: Control Effect of trans,trans-2,4-nonadienal and its inclusion complexes on postharvest anthracnose of loquat fruit.

[0065] Select loquat fruits of uniform size and ripeness, soak them in a 0.1% sodium hypochlorite solution for 10 minutes, rinse them twice with water, and then air dry them. 5 spores·mL -1 Soak loquat fruits in anthracnose spore solution for 10 minutes, then remove and air-dry for later use. Place 5 μL of trans,trans-2,4-nonadienal and 5 μL of the inclusion complex E24N / HPβCD-IC containing trans,trans-2,4-nonadienal in two sealed fruit boxes, respectively, as two experimental groups (E24N group and E24N / HPβCD-IC group); a control group (untreated fruit box) is also included. Place the air-dried loquat fruits into the three fruit boxes, then store the fruit boxes at 25℃. Observe and evaluate every 4 days according to the following criteria:

[0066] Level 1: No obvious browning caused by pathogens on the surface and no obvious wrinkling on the surface; Level 2: A small amount of browning caused by disease on the surface, less than 10% of the surface, and the surface is smooth without obvious wrinkling; Level 3: Browning caused by disease in multiple places on the surface, with the browned area reaching 10%-20%, and the surface is smooth without obvious wrinkling; Level 4: Browning caused by disease in multiple places on the surface, with the browned area reaching 10%-20%, and the surface showing signs of water loss; Level 5: Browning caused by disease in multiple places on the surface, with the browned area greater than 20%, and water seepage at the browned areas, but the surface is smooth without obvious wrinkling; Level 6: Browning caused by disease in multiple places on the surface, with the browned area greater than 20%, and water seepage at the browned areas, and the surface showing signs of water loss.

[0067] Disease incidence index = ∑(number of diseased fruits × disease grade) / (total number of fruits × highest disease grade) × 100%.

[0068] Rot rate = (Number of diseased fruits / Total number of fruits) × 100%.

[0069] The results are as follows Figure 8 As shown, the incidence rate in the control group was 32.22%, while the incidence rates in the E24N group and the E24N / HPβCD-IC group were 20% and 16.66%, respectively, both significantly lower than the control group. The rot rate in the control group was 44.44%, while the rot rates in the E24N group and the E24N / HPβCD-IC group were 33.33% and 27.56%, respectively, far lower than the control group. This indicates that trans, trans-2,4-nonadienal and its inclusion complex E24N / HPβCD-IC can delay the onset of postharvest anthracnose in loquat fruit, with the inclusion complex E24N / HPβCD-IC showing better efficacy.

[0070] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The application of trans,trans-2,4-nonadienal inclusion complex in the preparation of a drug for the prevention and treatment of loquat anthracnose, wherein loquat anthracnose is caused by *Anthracnose fusiforme*. Colletotrichum acutatum The anthracnose disease caused by the disease in loquat fruit; the trans, trans-2,4-nonadienal inclusion complex is an inclusion complex obtained by encapsulating trans, trans-2,4-nonadienal in hydroxypropyl-β-cyclodextrin; the molar ratio of trans, trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin is 1:

2.

2. The application as described in claim 1, characterized in that: The trans, trans-2,4-nonadienal inclusion complex can be prepared by the following method: S1. Dissolve hydroxypropyl-β-cyclodextrin in PBS buffer containing 20% ​​(mL / mL) ethanol at pH 6.8-7.2; S2. Dissolve trans,trans-2,4-nonadienal in anhydrous ethanol, then add it to the hydroxypropyl-β-cyclodextrin solution obtained in step S1 and mix well; the molar ratio of trans,trans-2,4-nonadienal to hydroxypropyl-β-cyclodextrin is 1:

2. S3. Sonicate the mixture obtained in step S2 at 65 W for 25-35 minutes; S4. Place the mixture after ultrasonic treatment in step S3 at 4℃ to 8℃ for 18-24 hours, filter it with a 0.2 μm microporous membrane, and collect the filtrate. S5. The filtrate obtained in step S4 is freeze-dried under vacuum at -50℃ to -60℃ for 36-48 h to obtain the inclusion complex.

3. The application as described in claim 2, characterized in that: In the preparation method of the trans, trans-2,4-nonadienal inclusion complex, the ratio of hydroxypropyl-β-cyclodextrin to PBS buffer is 0.1 mmol: 60 mL, and the ratio of trans, trans-2,4-nonadienal to anhydrous ethanol is 0.05 mmol: 2 mL; the ultrasonic treatment is carried out continuously for 30 minutes at 65 W power in a 1-second start-1-second pause mode.

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