Compound plant essential oil for inhibiting saprolegnia as well as preparation method and application of compound plant essential oil

Abstract

The invention relates to the technical field of aquaculture disease prevention and control, in particular to compound plant essential oil for inhibiting saprolegnia and a preparation method and application thereof.The compound plant essential oil comprises clove essential oil and cinnamon essential oil, and the mass ratio of the clove essential oil to the cinnamon essential oil is 1: (1-2). The compound plant essential oil disclosed by the invention has the advantages of wide action target, difficulty in generating drug resistance, no chemical residue and the like, and the stability and the safety of long-term use are ensured. Meanwhile, the special plant essential oil smell can effectively avoid stress reaction of aquatic animals caused by pungent smell of a traditional chemical bactericide, and the sustainable development requirement of the modern aquaculture industry is met.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture disease prevention and control, and in particular to a compound plant essential oil for inhibiting water molds, and a preparation method and application thereof. Background Art

[0002] Saprolegniasis is a common fungal parasitic disease in aquatic animal farming, primarily caused by Saprolegniae, such as S. ferax and S. parasitica. The disease is widespread in freshwater environments and can infect aquatic animals through physical injury or parasitic bites. It causes symptoms such as necrosis of the dermis and subcutaneous tissue, edema, degenerative changes in muscle fibers, and ulceration of the skin and fin rays. In severe cases, the mortality rate can reach as high as 100%, severely hindering the healthy development of the aquaculture industry.

[0003] Traditional methods for controlling saprolegniasis rely primarily on chemical antimicrobial agents and antibiotics, such as malachite green, which is now banned. However, long-term use not only leads to increasingly serious drug resistance but also poses potential risks to the environment and human health. Therefore, the development of novel, environmentally friendly, and harmless antifungal fishery drugs has become a research hotspot.

[0004] Plant essential oils are widely used in a variety of fields, including food, pharmaceuticals, and fragrances, due to their natural origins, diverse bioactivities, ease of absorption, and resistance to drug resistance. In recent years, research on the antibacterial, anti-inflammatory, and antioxidant properties of plant essential oils has continued to deepen, and they are considered potential alternatives to antibiotics. In aquaculture, in particular, the inhibitory effects of plant essential oils against key pathogens have garnered increasing attention. For example, cinnamaldehyde exhibits significant inhibitory effects against Staphylococcus aureus and Escherichia coli; oregano essential oil has excellent antibacterial activity against Vibrio vulnificus, a pathogen in marine aquaculture; and citronella essential oil has strong inhibitory activity against Vibrio and Edwardsiella. However, the antibacterial effects of individual essential oils are limited, and studies on combination essential oils against Saprolegnia have been rare. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a compound plant essential oil for inhibiting Saprolegnia, as well as a preparation method and application thereof, so as to solve the drug resistance problem and environmental pollution problem caused by the long-term use of chemical antibacterial agents and antibiotics in the prior art.

[0006] The present invention is achieved through the following technical solutions: on the one hand, a compound plant essential oil for inhibiting water molds is provided, wherein the compound plant essential oil comprises clove essential oil and cinnamon essential oil, wherein the mass ratio of the clove essential oil to the cinnamon essential oil is 1:1-2; preferably, the mass ratio of the clove essential oil to the cinnamon essential oil is 1:1.

[0007] Through the above-mentioned technical solution, while some of the currently reported plant essential oils have some inhibitory effect on Saprolegnia, the inhibitory effect of a single essential oil is often limited, making it difficult to achieve ideal control results. Research on plant essential oils targeting Saprolegnia is limited and lacks systematic and in-depth research. The synergistic effects and mechanisms between different essential oils are still unclear.

[0008] The present invention, based on the above technical solution and using the partial inhibitory concentration index (FICI) as a criterion, shows that when clove essential oil and cinnamon essential oil are mixed at a concentration of 1:1, they can effectively inhibit the growth of Saprolegnia spp., showing synergistic synergy and enhancing the antibacterial activity of the composite essential oil. The eugenol in clove essential oil has a unique phenolic ring structure that enables it to effectively bind to receptors on the cell wall of Saprolegnia spp., disrupting the integrity of the cell wall and causing leakage of cell contents, thereby inhibiting the growth of Saprolegnia spp. In addition, eugenol can further inhibit the reproduction of Saprolegnia spp. by affecting its metabolic pathways, such as inhibiting ATP synthesis and interfering with DNA replication.

[0009] Cinnamaldehyde, contained in cinnamon essential oil, has significant antifungal effects. For example, it has inhibitory effects on Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Pseudomonas fluorescens, Bacillus aeruginosa, and Microsporum gypseum. The aldehyde group in cinnamaldehyde makes it highly electrophilic, allowing it to react with biomacromolecules such as proteins and nucleic acids within Saprolegnia cells, leading to cell dysfunction and death. Furthermore, cinnamaldehyde can inhibit Saprolegnia growth by altering cell membrane permeability and disrupting its integrity. Therefore, the above-mentioned technical solution effectively addresses key issues hindering the sustainable development of the aquaculture industry, such as Saprolegnia resistance, drug residues, and environmental compatibility, providing an innovative solution for the prevention and control of Saprolegniasis in aquaculture.

[0010] A compound plant essential oil for inhibiting Saprolegniasis is based on the above compound plant essential oil for inhibiting Saprolegniasis. The compound plant essential oil comprises garlic essential oil, and the mass ratio of clove essential oil:cinnamon essential oil:garlic essential oil is 1:2-4:2-4; preferably, the mass ratio of clove essential oil:cinnamon essential oil:garlic essential oil is 1:2:2.

[0011] Through the above technical solution, the essential oil mixed with clove, cinnamon and garlic essential oils can effectively inhibit the growth of water mold with the minimum dosage.

[0012] The eugenol in clove essential oil has a unique phenolic ring structure that allows it to effectively bind to receptors on the cell walls of Saprolegnia spp., disrupting the integrity of the cell walls and causing leakage of cellular contents, thereby inhibiting the growth of the fungus. Furthermore, eugenol can further inhibit the growth of Saprolegnia spp. by affecting its metabolic pathways, such as inhibiting ATP synthesis and interfering with DNA replication.

[0013] Cinnamaldehyde, found in cinnamon essential oil, has significant antifungal effects. For example, it has inhibitory effects against Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Pseudomonas fluorescens, Bacillus aeruginosa, and Microsporum gypseum. The aldehyde group in cinnamaldehyde makes it highly electrophilic, allowing it to react with biomacromolecules such as proteins and nucleic acids within Saprolegnia cells, leading to cell dysfunction and death. Furthermore, cinnamaldehyde can inhibit Saprolegnia growth by altering cell membrane permeability and disrupting its integrity.

[0014] Allicin in garlic essential oil is a sulfur-containing compound with broad-spectrum antimicrobial activity. It reacts with thiol groups within Saprolegnia cells to form disulfide bonds, disrupting the intracellular redox balance and leading to cell death. Furthermore, allicin can inhibit Saprolegnia growth by affecting cell wall synthesis and membrane stability.

[0015] A compound plant essential oil for inhibiting water molds is based on the above compound plant essential oil for inhibiting water molds. The compound plant essential oil also includes oregano essential oil, and the mass ratio of clove essential oil: cinnamon essential oil: garlic essential oil: oregano essential oil is 1-2:1-2:1-4:2-4; preferably, the mass ratio of clove essential oil: cinnamon essential oil: garlic essential oil: oregano essential oil is 1-2:1-2:2:2.

[0016] Through the above technical solution, the cinnamaldehyde contained in cinnamon essential oil has significant antifungal effects. For example, it has inhibitory effects on Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Pseudomonas fluorescens, Bacillus aeruginosa, and Microsporum gypseum. The aldehyde group in cinnamaldehyde makes it highly electrophilic, allowing it to react with biomacromolecules such as proteins and nucleic acids within Saprolegnia cells, leading to cell dysfunction and death. Furthermore, cinnamaldehyde can inhibit the growth of Saprolegnia by altering cell membrane permeability and disrupting its integrity.

[0017] Allicin in garlic essential oil is a sulfur-containing compound with broad-spectrum antimicrobial activity. It reacts with thiol groups within Saprolegnia cells to form disulfide bonds, disrupting the intracellular redox balance and leading to cell death. Furthermore, allicin can inhibit Saprolegnia growth by affecting cell wall synthesis and membrane stability.

[0018] The main components of oregano essential oil, carvacrol and thymol, inhibit the growth of 20 fungi, including Fusarium, Aspergillus, and Penicillium. Furthermore, other terpenes, such as terpinene and α-terpineol, disrupt the cell membranes of Saprolegnias, causing leakage of cellular contents, affecting their metabolic pathways and inhibiting their growth. Furthermore, compared to traditional antibiotics, oregano essential oil is less likely to induce drug resistance against fungi.

[0019] The present invention selects four plant essential oils, including clove, cinnamon, garlic, and oregano, to compare their antibacterial effects on Saprolegnia spp., and selects the most potent essential oils for rational formulation. This formulation, through a multi-target mechanism of action, disrupts cell membrane integrity, inhibits ATP synthesis, and interferes with DNA synthesis. The combined system achieves potent inhibition of Saprolegnia spp. at low concentrations, increasing the antibacterial activity by 2-4 times compared to a single essential oil. Furthermore, the formulation is less susceptible to drug resistance and rapidly degrades in the environment, avoiding the ecological pollution caused by the use of chemical preparations, thus aligning with the development trend of green aquaculture.

[0020] Furthermore, it also includes a stabilizer, which is a pyrrolidone compound.

[0021] A method for preparing the above-mentioned compound plant essential oil for inhibiting Saprolegnia is also provided, comprising the following steps:

[0022] Step S1: weighing the compound plant essential oil according to the above mass ratio;

[0023] Step S2: dissolving the raw materials obtained in step S1 in a solvent, adding a stabilizer, and stirring evenly;

[0024] Step S3: Add deionized water to the mixture obtained in step S2 and stir evenly to obtain the product.

[0025] Furthermore, in step S2, the solvent is any one of tristyrylphenol polyoxyethylene ether, dimethyl sulfoxide, and propylene glycol.

[0026] Furthermore, in step S2, the stabilizer is polyvinyl pyrrolidone, and the added amount of the polyvinyl pyrrolidone accounts for 3% to 15% of the total amount of the plant essential oil solution.

[0027] Finally, the invention provides an application of the compound plant essential oil for inhibiting Saprolegnia in preventing and treating Saprolegniasis in aquaculture.

[0028] Furthermore, when the administration method is water treatment or fish immersion, the effective concentration is 3.12 to 25 μg / mL.

[0029] Beneficial effects

[0030] The present invention is based on the rational compounding of four essential oils and can achieve significant antibacterial effects at lower concentrations. The mechanism of action may be to exert the antibacterial effect by destroying the permeability of the cell membrane, the synthesis of the cell wall, or interfering with cell biosynthesis and energy metabolism.

[0031] Compared to chemically synthesized fungicides, the compound plant essential oils of this invention offer advantages such as a wide range of targets, low resistance to drug resistance, and no chemical residue, ensuring long-term stability and safety. Furthermore, the unique aroma of the plant essential oils effectively mitigates the stress reactions in aquatic animals caused by the pungent odor of traditional chemical fungicides, meeting the sustainable development requirements of the modern aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 MIC values ​​of single plant essential oils against water molds in vitro;

[0033] Figure 2 The antibacterial effect of compound plant essential oil on Saprolegnia;

[0034] Figure 3 The antibacterial effect of compound plant essential oil on Saprolegnia;

[0035] Figure 4 The antibacterial effect of compound plant essential oil on Saprolegnia;

[0036] Figure 5 Antibacterial effect of compound plant essential oils on Saprolegnia sp. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments herein without inventive effort are intended to fall within the scope of protection of the present invention. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise indicated, all percentages, ratios, proportions, and parts are by weight.

[0039] Unless otherwise specified, the reagents and raw materials used in the examples and comparative examples of the present invention can be obtained through commercial channels.

[0040] Example 1

[0041] A method for preparing a compound plant essential oil for inhibiting water molds, wherein the compound plant essential oil comprises clove essential oil and cinnamon essential oil;

[0042] The specific preparation steps are as follows:

[0043] Step S1: At room temperature, accurately weigh clove essential oil and cinnamon essential oil in a mass ratio of 1:1-2;

[0044] Step S2: dissolving the clove essential oil and cinnamon essential oil weighed in step S1 in a suitable solvent such as dimethyl sulfoxide or propylene glycol to ensure that the essential oils are completely dissolved;

[0045] Step S3: adding an appropriate amount of polyvinylpyrrolidone (PVP-K30) to the composite essential oil solution dissolved in step S2, and stirring evenly to obtain a stable mixture;

[0046] Step S4: adding the mixture obtained in step S3 to pretreated deionized water and stirring evenly to obtain the compound plant essential oil.

[0047] Test Example 1

[0048] 1. Antibacterial activity of single plant essential oils against Saprolegnia

[0049] Recovery of bacterial strains: After culturing the prepared PDA culture medium in a constant temperature incubator at 23°C for 24 hours, inoculate the rapeseed with bacterial strains into a culture dish on a clean bench and culture it in a constant temperature incubator at 23°C. Observe the growth of water molds every day. When the colony grows to 2 / 3 of the culture dish with a diameter of 90 mm, set it aside.

[0050] Prepare bacterial suspension: Aseptically, use an inoculating loop to pick Saprolegnia from the culture medium and place it on PDB medium. Incubate in a 23°C incubator. Observe and record the growth of Saprolegnia every 24 hours. Count the number of colonies using a hemocytometer and adjust the bacterial suspension concentration to 3 × 10⁵ to 5 × 10⁵ CFU / mL by adding deionized water.

[0051] The minimum inhibitory concentration (MIC) was determined using the broth microdilution method: PDB and the prepared essential oil were added to a 96-well plate. The final essential oil concentrations in each well were adjusted to 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 μg / mL, respectively, by two-fold dilution. Then, 100 μL of bacterial suspension was added. A positive control consisted of 200 μL of bacterial suspension, and a negative control consisted of 100 μL of dimethyl sulfoxide and 100 μL of PDB. The plates were incubated at 23°C for 24–48 hours. The MIC of the plant essential oil against Saprolegnia was observed and recorded.

[0052] Table 1 Antibacterial activity of single plant essential oils against Saprolegnia

[0053]

[0054]

[0055] As shown in Table 1, both clove essential oil and cinnamon essential oil can effectively inhibit the growth of Saprolegnia.

[0056] 2. In vitro antibacterial test of clove and cinnamon essential oils

[0057] Dilute clove oil and cinnamon oil to 1 / 2MIC and 1 / 4MIC respectively in an ELISA plate. Using the orthogonal experimental design method, pipette each solution. Finally, each well contains 100 μL of mixed essential oil and 100 μL of bacterial suspension. The negative control is 100 μL of single essential oil dilution and 100 μL of PDB, and the positive control is 200 μL of bacterial suspension. Incubate them in a constant temperature incubator at 23 °C for 24 - 48 h. Set 3 parallels for each group. If there is no bacteria growth in the well plate, it is recorded as the MIC value of the compound essential oil.

[0058] In this experiment, the fractional inhibitory concentration index (FICI) was used as the basis for judging the effect of the combined antibacterial test of essential oils. The judgment criteria are as follows:

[0059] FICI ≤ 0.5: Synergistic effect, that is, the effect of combined drug use is significantly better than that of single drug use.

[0060] 0.5 < FICI ≤ 1: Additive effect, that is, the effect of combined drug use is equal to the sum of the effects of single drugs.

[0061] 1 < FICI ≤ 2: Irrelevant effect, that is, the effect of combined drug use has no significant difference from that of single drug use.

[0062] FICI > 2: Antagonistic effect, that is, the effect of combined drug use is significantly lower than that of single drug use.

[0063] The calculation formula is as follows:

[0064] FICI = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone

[0065] Table 2 Antibacterial effect of compound plant essential oil against Saprolegnia

[0066]

[0067] Note: "+" indicates that there are colonies growing on the culture medium, and "-" indicates that there are no colonies on the culture medium.

[0068] As can be seen from Table 2, compared with the use of essential oils alone, when the mass ratio of clove oil to cinnamon oil is 1:1 - 1:2, there are no colonies growing on the culture medium. According to the FICI judgment criteria, when the mass ratio of clove oil to cinnamon oil is 1:1, it shows an additive effect. That is, the compound plant essential oil composed of clove oil and cinnamon oil with a mass ratio of 1:1 has a good inhibitory effect on Saprolegnia.

[0069] Experimental Example 2 Stability test of compound plant essential oil

[0070] Clove essential oil and cinnamon essential oil were accurately weighed and mixed in a 1:1 mass ratio. A solvent and a sustained-release agent were added to prepare a compound essential oil solution. The samples were divided into brown ampoules and placed in a constant temperature environment of -20℃, 25℃ and 40℃ for 30 days to observe whether the essential oil solution was stratified.

[0071] The results showed that the prepared compound plant essential oil showed excellent stability within the test temperature range of -20 to 40°C, and no stratification occurred.

[0072] Test Example 3 Safety Test of Compound Plant Essential Oil

[0073] A safety test was conducted on healthy grass carp (Ctenopharyngodon spp.) weighing 1-3g. The compound plant essential oil formulated in Experimental Example 12 was added to an aquarium containing these fish. During the test, the grass carp's behavior, body surface, and gills were observed. The safety of the compound plant essential oil was verified based on existing clinical experience with the product.

[0074] After observation, no pathological symptoms such as congestion and abnormal mucus secretion were found in the gill filaments and body surface of grass carp, and there were no abnormal behaviors such as wild swimming, rolling over, and rapid breathing.

[0075] Test Example 4 Clinical Effect Verification

[0076] 1. Fish immersion experiment

[0077] Healthy, one-year-old grass carp weighing 50-100g were artificially infected with Saprolegnia parasitica (ATCC 200013) provided by the National Aquatic Animal Pathogen Bank of Shanghai Ocean University. The animals were randomly divided into six blank groups and three control groups, with n = 20 per group, using a completely randomized block design. The experimental container was a standardized glass aquarium measuring 100 cm (L × W × H) by 50 cm (H) by 80 cm. The water parameters were maintained constant across all groups: pH 7.5-8.0, dissolved oxygen ≥ 5.0 mg / L, and temperature 23°C.

[0078] The compound plant essential oil determined in Test Example 12 was made into a medicament and added to the treatment group for medicated bathing, once a day for 5 to 7 consecutive days.

[0079] Observations showed that by the fifth day, 60% to 80% of the infected grass carp in the treated group had improved their skin lesions, with reduced white floccules, subsided redness and swelling, and improved feeding. By the seventh day, 80% of the infected grass carp had essentially healed their lesions and were feeding normally. In the control group, however, the mortality rate reached 30% on the fifth day, with 20% swimming sideways or lying on the bottom of the bathtub, refusing to eat. By the seventh day, the mortality rate had reached 60%.

[0080] Therefore, the compound plant essential oil composed of clove essential oil and cinnamon essential oil in a mass ratio of 1:1 has a significant inhibitory effect on water molds in aquatic animals and is non-toxic to aquatic animals.

[0081] 2. Water treatment experiment

[0082] Prepare the culture water containing water mold spores, divide it into 6 groups of blank groups and 3 groups of control groups, and the initial concentration of water mold in each group is controlled at 3×10 5 ~5×10 5 CFU / mL, and the physical and chemical parameters of the water in each group were maintained constant: pH 7.5-8.0, dissolved oxygen ≥5.0 mg / L, and temperature 23°C.

[0083] The compound plant essential oil determined in Test Example 12 was made into a medicament and added to the treatment group to verify the antibacterial effect. The number of viable Saprolegnia in the water body was detected every 6 hours, and the detection cycle was 24 hours.

[0084] The results showed that after 24 hours, the average number of viable Saprolegnia in the blank group reached 5×10 7 CFU / mL, while in the control group it was 5×10 4 The CFU / mL inhibition rate was 83%.

[0085] Therefore, the compound plant essential oil composed of clove essential oil and cinnamon essential oil in a mass ratio of 1:1 has a good inhibitory effect on Saprolegnia spores in water bodies and Saprolegnia in aquatic animals, with an inhibition rate of >80% within 24 hours, and is suitable for biological control in aquaculture water bodies.

[0086] Example 2

[0087] A method for preparing a compound plant essential oil for inhibiting water molds, wherein the compound plant essential oil comprises clove essential oil, cinnamon essential oil and garlic essential oil, and the specific preparation steps are as follows:

[0088] Step S1: At room temperature, accurately weigh clove essential oil, cinnamon essential oil, and garlic essential oil in a mass ratio of 1:2-4:2-4;

[0089] Step S2: dissolving the clove essential oil, cinnamon essential oil, and garlic essential oil weighed in step S1 in a suitable solvent such as dimethyl sulfoxide or propylene glycol, ensuring that the essential oils are completely dissolved;

[0090] Step S3: adding an appropriate amount of polyvinylpyrrolidone (PVP-K30) to the composite essential oil solution dissolved in step S2, and stirring evenly to obtain a stable mixture;

[0091] Step S4: adding the mixture obtained in step S3 to pretreated deionized water and stirring evenly to obtain the compound plant essential oil.

[0092] Test Example 1

[0093] 1. Antibacterial activity of single plant essential oils against Saprolegnia

[0094] The MIC value of garlic essential oil against Saprolegnia was determined according to the method in Test Example 11 of Example 1. The MIC value of garlic essential oil against Saprolegnia was 25.00 μg / mL.

[0095] 2. Combined in vitro antibacterial test of clove, cinnamon and garlic essential oils

[0096] According to the method in Example 1 and Test Example 12, the antibacterial effect of the compound plant essential oil on Saprolegnia was determined.

[0097] Table 3 Antibacterial effect of compound plant essential oil on Saprolegnia

[0098] Treatment group Clove essential oil Cinnamon essential oil Garlic essential oil Colony growth bacterial suspension 0 0 0 + 1 3.12 6.25 12.50 - 2 3.12 6.25 6.25 - 3 3.12 12.50 6.25 -

[0099] Note: “+” indicates that colonies have grown on the culture medium, and “-” indicates that no colonies have grown on the culture medium.

[0100] As shown in Table 3, clove essential oil, cinnamon essential oil and garlic essential oil can effectively inhibit the growth of Saprolegnia when mixed in mass ratios of 1:2:4, 1:2:2 and 1:4:2.

[0101] Test Example 2 Stability Test of Compound Plant Essential Oil

[0102] Clove, cinnamon, and garlic essential oils were precisely weighed and mixed in mass ratios of 1:2:4, 1:2:2, and 1:4:2, respectively. A solvent and a sustained-release agent were added to prepare a compound essential oil solution. The samples were dispensed into brown ampoules and stored at constant temperatures of -20°C, 25°C, and 40°C for 30 days. The essential oil solutions were then observed for stratification. The results showed that the formulated compound plant essential oil exhibited excellent stability within the tested temperature range of -20°C to 40°C, with no stratification observed.

[0103] Test Example 3 Safety Test of Compound Plant Essential Oil

[0104] A safety test was conducted on healthy grass carp (Ctenopharyngodon spp.) weighing 1-3 g. The compound plant essential oil formulated in Experimental Example 12 was added to an aquarium containing these fish. During the test, the grass carp's behavior, body surface, and gills were observed. The optimal concentration range of the compound plant essential oil was screened based on existing clinical experience with the product.

[0105] After observation, no pathological symptoms such as congestion and abnormal mucus secretion were found in the gill filaments and body surface of grass carp, and there were no abnormal behaviors such as wild swimming, rolling over, and rapid breathing.

[0106] Test Example 4 Clinical Effect Verification

[0107] 1 Fish immersion experiment

[0108] One-year-old healthy grass carp weighing 50-100 g was selected, and the compound plant essential oil determined in Test Example 12 was prepared into medicaments of different concentrations. The therapeutic effect of the compound plant essential oil was verified according to the experimental method in Example 1 and Test Example 41.

[0109] The results showed that the compound plant essential oil composed of clove essential oil, cinnamon essential oil and garlic essential oil in a mass ratio of 1:2-4:2-4 had a relieving and alleviating effect on the lesion sites of grass carp, and the feeding basically returned to normal on the 7th day.

[0110] 2 Water treatment experiment

[0111] The compound plant essential oil determined in Test Example 12 was prepared into pharmaceutical preparations of different concentrations, and the antibacterial effect in water was verified according to the experimental method in Example 1 and Test Example 42.

[0112] The results showed that the compound plant essential oil composed of clove essential oil, cinnamon essential oil and garlic essential oil in a mass ratio of 1:2-4:2-4 had a rapid killing effect on Saprolegnia spores, with a comprehensive inhibition rate of ≥85%.

[0113] Based on the comprehensive test results, the essential oil prepared by mixing clove essential oil, cinnamon essential oil and garlic essential oil in a mass ratio of 1:2:2 has more advantages in usage, therapeutic effect and water treatment, and can play a controlling role in the growth of Saprolegniasis and Saprolegniasis infection in aquatic animals.

[0114] Example 3

[0115] A method for preparing a compound plant essential oil for inhibiting water molds, wherein the compound plant essential oil comprises cinnamon essential oil, garlic essential oil and oregano essential oil, and the specific preparation steps are as follows:

[0116] Step S1: At room temperature, accurately weigh cinnamon essential oil, garlic essential oil, and oregano essential oil in a mass ratio of 1:1 to 2:2;

[0117] Step S2: dissolving the cinnamon essential oil, garlic essential oil, and oregano essential oil weighed in step S1 in a suitable solvent such as dimethyl sulfoxide or propylene glycol, ensuring that the essential oils are completely dissolved;

[0118] Step S3: adding an appropriate amount of polyvinylpyrrolidone (PVP-K30) to the composite essential oil solution dissolved in step S2, and stirring evenly to obtain a stable mixture;

[0119] Step S4: adding the mixture obtained in step S3 to pretreated deionized water and stirring evenly to obtain the compound plant essential oil.

[0120] Test Example 1

[0121] 1. Antibacterial activity of single plant essential oils against Saprolegnia;

[0122] The MIC value of oregano essential oil against Saprolegnia sp. was determined according to the method in Test Example 11 of Example 1. The MIC value of oregano essential oil against Saprolegnia sp. was determined to be 25.00 μg / mL.

[0123] 2. In vitro antibacterial test of cinnamon, garlic and oregano essential oils;

[0124] According to the method in Example 1 and Test Example 12, the antibacterial effect of the compound plant essential oil on Saprolegnia was determined.

[0125] Table 4 Antibacterial effect of compound plant essential oil on Saprolegnia

[0126] Treatment group Cinnamon essential oil Garlic essential oil Oregano essential oil Colony growth bacterial suspension 0 0 0 + 1 6.25 12.50 12.50 - 2 6.25 6.25 12.50 -

[0127] Note: “+” indicates that colonies have grown on the culture medium, and “-” indicates that no colonies have grown on the culture medium.

[0128] As shown in Table 4, when cinnamon essential oil, garlic essential oil and oregano essential oil are compounded in the mass ratio of 1:2:2 and 1:1:2, they can effectively inhibit the growth of Saprolegnia.

[0129] Test Example 2 Stability Test of Compound Plant Essential Oil

[0130] Cinnamon, garlic, and oregano essential oils were precisely weighed and mixed in a 1:2:2 and 1:1:2 mass ratio, and a solvent and sustained-release agent were added to prepare a compound essential oil solution. The samples were dispensed into brown ampoules and stored at constant temperatures of -20°C, 25°C, and 40°C for 30 days. The essential oil solutions were then observed for stratification. The results showed that the prepared compound plant essential oil exhibited excellent stability within the tested temperature range of -20°C to 40°C, with no stratification observed.

[0131] Test Example 3 Safety Test of Compound Plant Essential Oil

[0132] A safety test was conducted on healthy grass carp (Ctenopharyngodon spp.) weighing 1-3 g. The compound plant essential oil formulated in Experimental Example 12 was added to an aquarium containing these fish. During the test, the grass carp's behavior, body surface, and gills were observed. The optimal concentration range of the compound plant essential oil was screened based on existing clinical experience with the product.

[0133] After observation, no pathological symptoms such as congestion and abnormal mucus secretion were found in the gill filaments and body surface of grass carp, and there were no abnormal behaviors such as wild swimming, rolling over, and rapid breathing.

[0134] Test Example 4 Clinical Effect Verification

[0135] 1. Fish immersion experiment

[0136] One-year-old healthy grass carp weighing 50-100 g was selected, and the compound plant essential oil determined in Test Example 12 was prepared into medicaments of different concentrations. The therapeutic effect of the compound plant essential oil was verified according to the experimental method in Example 1 and Test Example 41.

[0137] The results showed that the compound plant essential oil composed of cinnamon essential oil, garlic essential oil and oregano essential oil in a mass ratio of 1:1 to 2:2 had a relieving and alleviating effect on the lesions of grass carp, and the feeding basically returned to normal on the 7th day.

[0138] 2. Water treatment experiment

[0139] The compound plant essential oil determined in Test Example 12 was prepared into pharmaceutical preparations of different concentrations, and the antibacterial effect in water was verified according to the experimental method in Example 1 and Test Example 42.

[0140] The results showed that the two groups of composite essential oil combinations with different concentrations had a rapid killing effect on Saprolegnia spores, with a comprehensive inhibition rate of ≥80%.

[0141] According to the comprehensive test results, the compound plant essential oil composed of cinnamon essential oil, garlic essential oil and oregano essential oil in a mass ratio of 1:1 to 2:2 has a good inhibitory effect on water treatment and water mold in aquatic animals.

[0142] Example 4

[0143] A method for preparing a compound plant essential oil for inhibiting water molds, wherein the compound plant essential oil comprises clove essential oil, cinnamon essential oil, garlic essential oil and oregano essential oil, and the specific preparation steps are as follows:

[0144] Step S1: At room temperature, accurately weigh clove essential oil, cinnamon essential oil, garlic essential oil, and oregano essential oil in a mass ratio of 1-2:1-2:1-4:2-4;

[0145] Step S2: dissolving the clove essential oil, cinnamon essential oil, garlic essential oil, and oregano essential oil weighed in step S1 in a suitable solvent such as dimethyl sulfoxide or propylene glycol, ensuring that the essential oils are completely dissolved;

[0146] Step S3: adding an appropriate amount of polyvinylpyrrolidone (PVP-K30) to the composite essential oil solution dissolved in step S2, and stirring evenly to obtain a stable mixture;

[0147] Step S4: adding the mixture obtained in step S3 to pretreated deionized water and stirring evenly to obtain the compound plant essential oil.

[0148] Test Example 1

[0149] (1) Antibacterial effect of compound plant essential oil on Saprolegnia

[0150] Clove, cinnamon, garlic, and oregano essential oils were diluted twofold to 1 / 2, 1 / 4, and 1 / 8 MIC in an ELISA plate. Using an orthogonal design, the individual essential oils were mixed, resulting in each well containing 100 μL of the combined essential oils and 100 μL of bacterial suspension. The optimal concentration range of the compound essential oils was determined based on the growth of Saprolegnia fasciculata.

[0151] Table 5 Antibacterial effect of compound plant essential oil on Saprolegnia

[0152] Formula Clove essential oil Cinnamon essential oil Garlic essential oil Oregano essential oil Colony growth 1 1 / 2MIC 1 / 2MIC 1 / 2MIC 1 / 2MIC - 2 1 / 2MIC 1 / 4MIC 1 / 4MIC 1 / 4MIC - 3 1 / 2MIC 1 / 4MIC 1 / 8MIC 1 / 4MIC + 4 1 / 4MIC 1 / 2MIC 1 / 4MIC 1 / 4MIC - 5 1 / 4MIC 1 / 4MIC 1 / 2MIC 1 / 2MIC + 6 1 / 4MIC 1 / 8MIC 1 / 8MIC 1 / 8MIC + 7 1 / 8MIC 1 / 4MIC 1 / 4MIC 1 / 8MIC + 8 1 / 8MIC 1 / 8MIC 1 / 4MIC 1 / 4MIC + 9 1 / 8MIC 1 / 4MIC 1 / 8MIC 1 / 8MIC +

[0153] Note: “+” indicates that colonies have grown in the culture medium, and “-” indicates that no colonies have appeared in the culture medium.

[0154] As shown in Table 4, formulas 1, 2 and 4 can effectively inhibit the growth of Saprolegnia.

[0155] Test Example 2 Stability Test of Compound Plant Essential Oil

[0156] Clove essential oil, cinnamon essential oil, garlic essential oil and oregano essential oil were accurately weighed and mixed in a mass ratio of 1-2:1-2:2:2, and a solvent and a sustained-release agent were added to prepare a compound essential oil solution. The samples were divided into brown ampoules and placed in a constant temperature environment of -20℃, 25℃ and 40℃ for a storage period of 30 days to observe whether the essential oil solution was stratified.

[0157] The results showed that the prepared compound plant essential oil showed excellent stability within the test temperature range of -20 to 40°C, and no stratification occurred.

[0158] Test Example 3 Safety Test of Compound Plant Essential Oil

[0159] A safety test was conducted on healthy grass carp (Ctenopharyngodon spp.) weighing 1-3 g. The compound plant essential oil prepared in (1) was added to the aquarium of grass carp (Ctenopharyngodon spp.). During the test, the behavior, body surface, and gill status of the grass carp were observed. The optimal concentration range of the compound plant essential oil was screened based on the experience of using existing clinical products.

[0160] After observation, no pathological symptoms such as congestion and abnormal mucus secretion were found in the gill filaments and body surface of grass carp, and there were no abnormal behaviors such as wild swimming, rolling over, and rapid breathing.

[0161] Test Example 4 Clinical Effect Verification

[0162] 1. Fish immersion experiment

[0163] One-year-old healthy grass carp weighing 50-100 g was selected, and the compound plant essential oil determined in 1 was prepared into a medicament of different concentrations. The therapeutic effect of the compound plant essential oil was verified according to the experimental method in Example 1 and Test Example 41.

[0164] The results showed that the compound plant essential oil composed of clove essential oil, cinnamon essential oil, garlic essential oil and oregano essential oil in a mass ratio of 1-2:1-2:2:2 had a relieving and alleviating effect on the lesions of grass carp. On the 7th day, the feeding basically returned to normal. If the use time was continued to 10 days, the cure rate of grass carp infected with Saprolegniasis reached 90%.

[0165] 2. Water treatment experiment

[0166] The compound plant essential oil determined in Test Example 12 was prepared into pharmaceutical preparations of different concentrations, and the antibacterial effect in water was verified according to the experimental method in Example 1 and Test Example 42.

[0167] The results showed that the two groups of composite essential oil combinations with different concentrations had a rapid killing effect on Saprolegnia spores, with a comprehensive inhibition rate of ≥90%.

[0168] Therefore, the systematic comparative analysis based on Test Examples 1 to 4 shows that in this embodiment, the compound preparation of clove essential oil, cinnamon essential oil, garlic essential oil and oregano essential oil configured in a mass ratio of 1 to 2:1 to 2:2:2 exhibits a significant inhibitory effect against water molds in aquatic animals.

[0169] In summary, the present invention has developed a synergistic anti-water mold composition based on clove essential oil, cinnamon essential oil, garlic essential oil, and oregano essential oil through systematic screening and scientific compounding. This effectively solves key issues such as water mold resistance, drug residues, and environmental compatibility that restrict the sustainable development of the aquaculture industry, and provides an innovative solution for the prevention and control of water mold disease in aquaculture. Compared with the existing technology, this formula destroys cell membrane integrity, inhibits ATP synthesis, interferes with DNA synthesis, etc. through a multi-target mechanism of action. Its compound system can achieve a strong inhibitory effect on water molds at low concentrations. The 24h antibacterial rate of the composite plant essential oil in Examples 1 to 4 is greater than 80%, which is 2-4 times higher than the antibacterial activity of a single essential oil. At the same time, it is not easy to develop drug resistance and can be rapidly degraded in the environment, avoiding the ecological pollution caused by the use of chemical preparations, which is in line with the development trend of green aquaculture.

[0170] This invention can be directly used in aquaculture water treatment, fish soaking, and other applications, and is suitable for aquatic animals susceptible to Saprolegniasis. Its natural properties can also be combined with probiotics and immunopotentiators to establish a comprehensive prevention and control system, promoting the sustainable development of the aquaculture industry.

[0171] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound plant essential oil for inhibiting water mold, characterized in that: The compound plant essential oil comprises clove essential oil and cinnamon essential oil, and the mass ratio of the clove essential oil to the cinnamon essential oil is 1:1-2.

2. A compound plant essential oil for inhibiting Saprolegnia, characterized in that: The compound plant essential oil for inhibiting water mold according to claim 1 comprises garlic essential oil, and the mass ratio of clove essential oil: cinnamon essential oil: garlic essential oil is 1:2-4:2-4.

3. A compound plant essential oil for inhibiting water mold, characterized in that: Based on the compound plant essential oil for inhibiting water molds according to claim 2, the compound plant essential oil also includes oregano essential oil, and the mass ratio of clove essential oil: cinnamon essential oil: garlic essential oil: oregano essential oil is 1-2:1-2:2:

2.

4. The compound plant essential oil for inhibiting Saprolegnia according to any one of claims 1 to 3, wherein The invention also includes a stabilizer, which is a pyrrolidone compound.

5. A method for preparing the compound plant essential oil for inhibiting Saprolegnia according to claim 4, characterized in that: The following steps are involved: Step S1: weighing the compound plant essential oil according to the above mass ratio; Step S2: dissolving the raw materials obtained in step S1 in a solvent, adding a stabilizer, and stirring evenly; Step S3: Add deionized water to the mixture obtained in step S2 and stir evenly to obtain the product.

6. The method for preparing the compound plant essential oil for inhibiting Saprolegnia according to claim 5, wherein In step S2, the solvent is any one of tristyrylphenol polyoxyethylene ether, dimethyl sulfoxide, and propylene glycol.

7. The method for preparing the compound plant essential oil for inhibiting Saprolegnia according to claim 5, wherein In step S2, the stabilizer is polyvinyl pyrrolidone, and the added amount of the polyvinyl pyrrolidone accounts for 3% to 15% of the total amount of the plant essential oil solution.

8. Use of the compound plant essential oil for inhibiting Saprolegnia according to any one of claims 1 to 3 for preventing and treating Saprolegniasis in aquaculture.

9. The use according to claim 8, characterized in that When the application method is water treatment or fish immersion, the effective concentration is 3.12-25 μg / mL.

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