Applications of indocalamus leaf essential oil and active components thereof in inhibition of aspergillus flavus

Abstract

The invention relates to the technical field of plant source mildew preventive, in particular to application of indocalamus leaf essential oil and active ingredients thereof in inhibition of aspergillus flavus. Experiments prove that the indocalamus leaf essential oil has the functions of inhibiting aspergillus flavus growth and toxin production, main active substances in the indocalamus leaf essential oil are further obtained through gas chromatography-mass spectrometry, and the main active substances linalool and longifolene are further verified to inhibit the aspergillus flavus growth and toxin production activity. Powerful experimental support is provided for research and development of a novel plant source mildew preventive.

Description

Technical Field

[0001] This invention relates to the field of plant-derived antifungal agents, and in particular to the application of bamboo leaf essential oil and its active ingredients in inhibiting Aspergillus flavus. Background Art

[0002] Aspergillus flavus is an aerobic saprophytic fungus that can contaminate almost all agricultural products, including wheat, rice, corn, peanuts, nuts, and cottonseed, producing the toxic secondary metabolite aflatoxin. Aflatoxin B1 is the most potent naturally occurring carcinogen and is classified as a Group 1A carcinogen by the International Agency for Research on Cancer (IARC). Long-term consumption of aflatoxin-contaminated food can seriously harm human and animal health and may cause cancer. Therefore, developing effective strategies to control Aspergillus flavus and aflatoxin contamination is extremely important. Currently, various technologies for controlling aflatoxin and its toxins have been developed, including the cultivation of disease-resistant varieties, temperature and humidity control, chemical fungicides, biological fungicides, and plant-derived fungicides. Among these, chemical fungicides are the most common and effective, but they have drawbacks such as high toxicity, environmental pollution, and damage to nutrients. Compared with traditional chemical fungicides, plant-derived fungicides have advantages such as being non-toxic, highly effective, having no side effects, being environmentally friendly, and not causing drug resistance, making them more suitable for the food industry.

[0003] Besides providing a unique flavor and aroma to zongzi (sticky rice dumplings), bamboo leaves also contribute to the longer shelf life of zongzi packaged in bamboo leaves, suggesting a certain degree of anti-corrosion ability. Chemical analysis shows that bamboo leaf extract contains various molecules, such as polysaccharides, phenolic acids, flavonoids, anthraquinones, coumarins, specific peptides, and volatile components. Furthermore, bamboo leaves possess biological activities such as anti-stress, anti-cancer, anti-inflammatory, and anti-cardiovascular disease effects. Therefore, bamboo leaves are a high-quality plant resource. However, the antifungal activity of bamboo leaf essential oil and its main active ingredients have never been reported. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems, the following invention is proposed.

[0005] In a first aspect, the present invention provides the use of bamboo leaf essential oil in at least the following aspects: (1) Inhibits the growth of Aspergillus flavus; (2) Inhibits the germination of Aspergillus flavus spores; (3) Inhibit Aspergillus flavus from producing aflatoxin.

[0006] Secondly, the present invention provides the use of linalool in at least one of the following aspects: (1) Inhibits the growth of Aspergillus flavus; (2) Inhibit Aspergillus flavus from producing aflatoxin.

[0007] Thirdly, the present invention provides the application of longifolene in inhibiting the production of aflatoxin by Aspergillus flavus.

[0008] Fourthly, the present invention provides the application of bamboo leaf essential oil in the preparation of products; said products are used in at least one of the following aspects: (1) Inhibits the growth of Aspergillus flavus; (2) Inhibits the germination of Aspergillus flavus spores; (3) Inhibit Aspergillus flavus from producing aflatoxin.

[0009] Fifthly, the present invention provides the use of linalool in the preparation of products; said products are used in at least one of the following aspects: (1) inhibiting the growth of Aspergillus flavus; (2) Inhibit Aspergillus flavus from producing aflatoxin.

[0010] In a sixth aspect, the present invention provides the application of longifolene in the preparation of products; said products are used to inhibit the production of aflatoxin by Aspergillus flavus.

[0011] Preferably, the product is a pharmaceutical, preservative, disinfectant, bactericide, or antifungal agent.

[0012] Preferably, the aflatoxin is aflatoxin B1.

[0013] Preferably, the main components of the bamboo leaf essential oil include: longleafene, linalool, 6-isopropylidene-1-methyl-bicyclohexane, 2-docosyl ketone, α-pine acetate, caryophyllene, E-cinnamaldehyde, β-myrcene, D-limonene, and 2-methoxy-3-(2-propenyl)-phenol.

[0014] Preferably, the essential oil of *Rubus umbellatus* leaves mainly comprises the following substances in weight percentage: 9.5%–9.7% longifolene, 7.9%–8% linalool, 5%–5.5% 6-isopropylidene-1-methyl-bicyclohexane, 4.5%–5% 2-docosyl ketone, 4%–5% α-pine acetate, 4%–5% caryophyllene, 4%–4.5% E-cinnamaldehyde, 3.5%–4% β-myrcene, 2.5%–3% D-limonene, and 2.5%–3% 2-methoxy-3-(2-propenyl)-phenol.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention experimentally confirmed that the essential oil of *Ipomoea aquatica* leaves has the function of inhibiting the growth and toxin production of *Aspergillus flavus*. Furthermore, the main active substances in the essential oil of *Ipomoea aquatica* leaves were obtained through gas chromatography-mass spectrometry analysis, and the inhibitory activities of the main active substances linalool and longifolene on the growth and toxin production of *Aspergillus flavus* were further verified, providing strong experimental support for the development of new plant-derived antifungal agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the test results of how bamboo leaf essential oil inhibits the growth and activity of Aspergillus flavus.

[0017] Figure 2 These are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of Aspergillus flavus treated with bamboo leaf essential oil.

[0018] Figure 3 This is a gas chromatography-mass spectrometry (GC-MS) analysis result of the essential oil from bamboo leaves.

[0019] Figure 4 This is a graph showing the antifungal activity test results of the main active ingredients, longifolene and linalool, in bamboo leaf essential oil.

[0020] Figure 5 This is a graph showing the test results of the effect of bamboo leaf essential oil on the control of aflatoxin in corn. DETAILED DESCRIPTION

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0022] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.

[0023] The bamboo leaf essential oil in the following examples was purchased from Jiangxi Gannan Natural Fragrance Oil Co., Ltd., item number 0254411; the longifole essential oil was purchased from Shanghai Yaoyao Fragrance Co., Ltd., item number 475-20-7; and the linalool essential oil was purchased from Jiangxi Xuesong Natural Medicinal Oil Co., Ltd., item number 2018120329.

[0024] Example 1: Determination of the inhibitory activity of bamboo leaf essential oil against Aspergillus flavus.

[0025] This embodiment uses a fumigation inhibition method to determine the anti-aspergillus activity of *Ipomoea aquatica* leaf essential oil. The specific steps are as follows: 10 μL of *Ipomoea aquatica* leaf essential oil was dropped onto a small piece of filter paper, which was then attached to the lid of a petri dish. At 28°C, 10 μL aliquots of *Aspergillus flavus* spore suspension were spotted onto PDA and YES petri dishes, respectively. Similarly, different amounts of *Ipomoea aquatica* leaf essential oil (0, 2, 4, 6, 8, and 10 μL) were added to the petri dish lids to detect the inhibition efficiency. After 7 days of incubation, the diameter of *Aspergillus flavus* colonies was measured to determine the inhibitory effect of different treatment concentrations on *Aspergillus flavus* growth. Simultaneously, *Aspergillus flavus* conidia were collected from the PDA plate, and the number of conidia was counted using a hemocytometer. Aflatoxin B1 was extracted from different *Ipomoea aquatica* leaf essential oil treatment groups from the YES plate. For aflatoxin extraction, five agar plugs containing mycelia were extracted with 5 mL of methanol, centrifuged, and purified using an immunoaffinity column. The aflatoxin was then detected by HPLC (high performance liquid chromatography). The HPLC conditions included: an Agilent HC-C18 column (4.6 mm × 250 mm, 5 μm), a mobile phase of 70% methanol, a detection time of 10 min, a flow rate of 1 mL / min, an injection volume of 10 μL, a column temperature of 30 °C, and excitation and emission wavelengths of 360 nm and 440 nm, respectively.

[0026] The results are as follows Figure 1 As shown, the inhibition rate of Aspergillus flavus growth significantly increased with increasing concentration of *Rubus idaeus* leaf essential oil. 10 μL of *Rubus idaeus* leaf essential oil completely inhibited the growth of *Aspergillus flavus* on PDA and YES media. Figure 1 A). Bamboo leaf essential oil can also significantly inhibit Aspergillus flavus spore germination and aflatoxin B1 production; 6 μL of bamboo leaf essential oil can completely inhibit Aspergillus flavus germination on PDA medium. Figure 1 B), 10 μL of *Ruzhu* leaf essential oil can completely inhibit the production of toxins by *Aspergillus flavus* on YES medium. Figure 1 C).

[0027] Furthermore, this embodiment uses scanning electron microscopy and transmission electron microscopy to detect the effects of bamboo leaf essential oil on Aspergillus flavus cells. The specific steps are as follows: Scanning electron microscopy: 4 μL of *Rubus idaeus* leaf essential oil was added to the lid of a petri dish and inoculated with *Aspergillus flavus* spores. The mixture was incubated at 28°C for 5 days. The *Aspergillus flavus* mycelium samples were then cut into 0.1–0.2 mm thick agar plugs. The cut mycelium was mixed with formaldehyde, washed with 0.2 M PBS buffer (pH 7.4), and dehydrated using a gradient of ethanol solutions (30, 60, 80, 90, maintaining 100%). The samples were then spot-dried, coated with gold sputtering, and finally the effects of *Rubus idaeus* leaf essential oil on *Aspergillus flavus* hyphae and spores were observed using a Hitachi S750 scanning electron microscope.

[0028] Transmission electron microscopy (TEM): Cut Aspergillus flavus cakes were mixed with 2.5% glutaraldehyde, washed twice with 0.2M PBS buffer (pH 7.4), fixed with 1% osmium tetroxide for 2 hours, and then washed again with PBS. The samples were then dehydrated in a fractionated ethanol series, passed through acetone-ethanol solution three times, and embedded in epoxy resin at 65°C for 16 hours. Finally, the samples were cut into 85 nm ultrathin sections using a diamond scalpel and stained with uranyl acetate and lead citrate. The effects of *Ipomoea aquatica* leaf essential oil on the cell structure of *Aspergillus flavus* were observed using an H-7500 TEM.

[0029] The results of scanning electron microscopy and transmission electron microscopy are as follows: Figure 2 As shown, in the control group, the Aspergillus flavus mycelium was straight and uniform, with a smooth surface without pitting, plump and undamaged conidial heads, straight conidia, and a large number of conidia were easily observed in the field of view. Figure 2 In treatment groups A and 2C), the mycelium was wrinkled, slender, and curled, with a rough and wrinkled surface. The conidial heads were atrophied and abnormally developed, the conidia were atrophied and twisted, and the number of conidia was significantly reduced. Figure 2 (B and 2D).

[0030] Transmission electron microscopy results showed that the control group of Aspergillus flavus cells exhibited a healthy structure, with a regular cell wall, cell membrane, and various organelles. Figure 2 E). The cell walls of *Aspergillus flavus* in the treatment group showed uneven thickness, blurred and wrinkled cell membranes, and significant changes in organelles, including blurred cytoplasm, collapsed and ruptured plasma membrane structures, and leakage of vacuolar exudate, especially an abnormal increase in mitochondria and lipid droplets. Figure 2 F).

[0031] Scanning electron microscopy and transmission electron microscopy results showed that the essential oil of bamboo leaves inhibited the growth and toxin production of Aspergillus flavus by disrupting the mycelial and cellular structures of Aspergillus flavus.

[0032] Example 2: Gas Chromatography-Mass Spectrometry Detection of Major Active Substances in Bamboo Leaf Essential Oil

[0033] This embodiment utilizes a TRACE 1310 gas chromatograph and an RTX-5MS flexible quartz capillary column for gas chromatography-mass spectrometry (GC-MS) analysis of *Imperata cylindrica* leaf essential oil. The initial column temperature was maintained at 50°C for 1 minute, then increased to 250°C at a rate of 10°C / min. The evaporation chamber temperature was 250°C, and the injection volume was 1 μL. Helium was used as the carrier gas at a flow rate of 1 mL / min and a split ratio of 50:1. MS analysis was performed using a TSQ 9000 triple-quadruple mass spectrometer in electron collision mode at 250°C, with an electron energy of 70 eV, a detection voltage of 0.8 kV, and a mass range of 20–550 amu. MS data were matched with NIST library data to identify volatile compounds. The relative peak areas were used to quantify different volatile compounds.

[0034] Table 1 lists the individual chemical substances with relative peak areas in *Rhododendron molle* leaf essential oil. A total of 166 chemical substances were identified by GC-MS and matched against an MS database; these substances accounted for 83.86% of the components in *Rhododendron molle* leaf essential oil. Further investigation of the top 70 components revealed that alkenes accounted for the largest proportion (26.99%), followed by alcohols (22.36%), esters (6.65%), alkanes (6.37%), acids (5.15%), ketones (4.23%), aldehydes (3.25%), naphthalenes (1.74%), oximes (1.72%), indenes (0.99%), benzyl groups (0.61%), hydrazines (0.56%), hydrocarbons (0.30%), and ethers (0.11%). Figure 3 A). The top ten compounds ( Figure 3 B) was identified as 9.55% longifolene ( Figure 3 C), 7.95% linalool ( Figure 3 D), 5.10% 6-isopropylidene-1-methyl-bicyclohexane, 4.80% 2-docosyl ketone, 4.44% α-acetate, 4.43% caryophyllene, 4.01% E-cinnamaldehyde, 3.74% β-myrcene, 2.94% D-limonene, and 2.92% 2-methoxy-3-(2-propenyl)phenol.

[0035] Table 1. Main active ingredients in bamboo leaf essential oil

[0036] Example 3: Verification of the antifungal activity of active ingredients in bamboo leaf essential oil

[0037] To verify the antifungal activity of longifolene and linalool, different amounts of longifolene and linalool essential oils (0, 2, 4, 6, 8, and 10 μL) were dropped onto a small filter paper, which was then attached to the petri dish lid. 10 μL of Aspergillus flavus spore suspension was then spotted onto PDA and YES petri dishes. After incubation at 28°C for 7 days, the diameter of Aspergillus flavus colonies was measured to determine the inhibitory effect of different treatment concentrations on Aspergillus flavus growth. Aflatoxin B1 was extracted from the YES plates from the different longifolene and linalool essential oil treatment groups. For aflatoxin extraction, five agar plugs containing mycelia were extracted with 5 mL of methanol. The extracts were centrifuged and purified using an immunoaffinity column, followed by HPLC (high performance liquid chromatography) detection under the same conditions as in Example 1.

[0038] The results are as follows Figure 4As shown, different concentrations of longleafene had no significant inhibitory effect on the growth of Aspergillus flavus. Figure 4 A and 4B), but when the concentration of longifolene was increased to 4 μL, the yield of AFB1 decreased significantly. When Aspergillus flavus was treated with 10 μL of longifolene, the yield of aflatoxin B1 was less than 1 / 3 of the control. Figure 4 C). Compared to linalool, linalool exhibited a stronger inhibitory effect on Aspergillus flavus. When the amount of linalool exceeded 4 μL per dish, the mycelial growth of Aspergillus flavus was significantly inhibited. Figure 4 D and 4E). Similarly, linalool treatment significantly reduced the level of aflatoxin B1 produced by Aspergillus flavus, and the 10 μL linalool treatment group completely inhibited the production of aflatoxin B1. Figure 4 F). It is evident that longifolene and linalool, as the main components of bamboo leaf essential oil, both exhibit the ability to inhibit the production of aflatoxin B1 by Aspergillus flavus.

[0039] Example 4: The Control Effect of Bamboo Leaf Essential Oil on Aspergillus Aflatoxin in Corn

[0040] This embodiment uses the fumigation inhibition method to determine the control effect of *Ipomoea auricula-judae* leaf essential oil on *Aspergillus flavus* in corn. The specific steps are as follows: 10 μL of *Ipomoea auricula-judae* leaf essential oil was dropped onto a small filter paper, which was then attached to the lid of a petri dish. 10 g of corn inoculated with *Aspergillus flavus* spores was evenly spread at the bottom of the petri dish and cultured at 28℃ for 7 days. The *Aspergillus flavus* infection status in the corn was observed, and the aflatoxin B1 content in the corn was determined according to the method in Example 1. For aflatoxin extraction, 50 mL of methanol was mixed with the corn in the petri dish for 30 min, centrifuged at 8000 rpm, and the supernatant was collected. The supernatant was purified using an immunoaffinity column and then detected by HPLC (high performance liquid chromatography) under the same conditions as in Example 1.

[0041] The results are as follows Figure 5 As shown, after 7 days of storage, visible Aspergillus flavus appeared on the corn in the control group. Figure 5 A) Most of the corn was infected with Aspergillus flavus, while the corn in the control group (treatment group) was not infected with Aspergillus flavus. Figure 5 B). Detection of aflatoxin B1 content in corn showed that the aflatoxin B1 content in the control group corn was 0.17 μg / mL, far exceeding the national limit of 0.02 μg / mL, while no aflatoxin B1 was detected in the control group. Figure 5 C).

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Indocalamus leaf essential oil in at least one of the following aspects: (1) Inhibit the growth of Aspergillus flavus; (2) Inhibit the germination of Aspergillus flavus spores; (3) Inhibit the production of aflatoxin by Aspergillus flavus.

2. Application of linalool in at least one of the following aspects: (1) Inhibit the growth of Aspergillus flavus; (2) Inhibit the production of aflatoxin by Aspergillus flavus.

3. The application of longifolene in inhibiting the production of aflatoxin by Aspergillus flavus.

4. Application of Indocalamus leaf essential oil in preparing products; the products are used for at least one of the following aspects: (1) Inhibit the growth of Aspergillus flavus; (2) Inhibit the germination of Aspergillus flavus spores; (3) Inhibit the production of aflatoxin by Aspergillus flavus.

5. Use of linalool in preparing products; the product is used in at least one of the following aspects: (1) Inhibit the growth of Aspergillus flavus; (2) Inhibit the production of aflatoxin by Aspergillus flavus.

6. Application of longifolene in the preparation of products; the products are used to inhibit Aspergillus flavus from producing aflatoxin.

7. The use according to any one of claims 4 to 6, characterized in that: The product is a medicine, a preservative, a disinfectant, a bactericide or a mildewcide.

8. The use according to any one of claims 1 to 7, characterized in that: The aflatoxin is aflatoxin B1.

9. The use according to claim 1, characterized in that: The main components of the indocalamus leaf essential oil include: longifolene, linalool, 6-isopropylidene-1-methyl-bicyclohexane, 2-docosanone, α-pinene acetate, caryophyllene, E-cinnamaldehyde, β-myrcene, D-limonene and 2-methoxy-3-(2-propenyl)-phenol.

10. The use according to claim 9, characterized in that: The indocalamus leaf essential oil mainly comprises the following substances in percentage by weight: 9.5% to 9.7% of longifolene, 7.9% to 8% of linalool, 5% to 5.5% of 6-isopropylidene-1-methyl-bicyclohexane, 4.5% to 5% of 2-docosanone, 4% to 5% of α-pinene acetate, 4% to 5% of caryophyllene, 4% to 4.5% of E-cinnamaldehyde, 3.5% to 4% of β-myrcene, 2.5% to 3% of D-limonene and 2.5% to 3% of 2-methoxy-3-(2-propenyl)-phenol.

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