Preparation of cinnamomum camphora essential oil nano-emulsion and application of cinnamomum camphora essential oil nano-emulsion as fish anesthetic in water-containing live-keeping transportation method of sea bass

Abstract

The invention discloses preparation of a cinnamomum camphora essential oil nano-emulsion and application of the cinnamomum camphora essential oil nano-emulsion as a fish anesthetic in a sea bass live-keeping transportation method in water, which comprises the following steps: S1, dissolving 5mL of Tween 80 in a deionized water solution by using Tween 80 as an emulsifier, and magnetically stirring at room temperature for 60 minutes to prepare a Tween 80 emulsifier; step S2, adding 5g of cinnamomum camphora essential oil into the stock solution subjected to magnetic stirring in the step S1, and performing high-speed homogeneous stirring for 10 minutes to obtain a cinnamomum camphora essential oil crude emulsion with the concentration of 50 mg / mL; s3, the crude emulsion is subjected to ultrasonic treatment, the power density of ultrasonic treatment is 573.75 W / cm < 2 >, the frequency is 20 kHz, the time is 30 min, and finally the nanometer emulsion of the cinnamomum camphora essential oil is prepared. The cinnamomum camphora nano-emulsion provided by the invention can eliminate the problems of poor solubility of essential oil in water and side effects of ethanol, and can achieve shorter anesthesia and recovery time by using a lower concentration.

Description

Technical Field

[0001] The invention relates to the technical field of nanoemulsion preparation, in particular to the preparation of a camphor essential oil nanoemulsion and application of the nanoemulsion as a fish anesthetic in a method for keeping sea bass alive in water. Background Art

[0002] The roots, branches, leaves, and fruits of Cinnamomum camphora are all used medicinally, with analgesic, anti-inflammatory, expectorant, cough-relieving, and wound-healing properties. Camphor and camphor oil, extracted from its wood, roots, branches, and leaves, are widely used as raw materials in the food, chemical, pharmaceutical, and fragrance industries. Camphor essential oil, primarily containing terpinene, cinnamyl acetate, isolinaol, and limonene, exhibits antibacterial, anticancer, antioxidant, and anti-inflammatory properties. The main components of camphor essential oil are α-terpineol, 2-camphenol (bornyl), and 1,8-cineole, most of which are monoterpenoid derivatives. Staphylococcus aureus and Escherichia coli are relatively sensitive to camphor essential oil, while Aspergillus niger and Penicillium are less sensitive. The mechanism by which Staphylococcus aureus responds to camphor essential oil is that the essential oil disrupts the permeability of the S. aureus cell membrane, allowing its contents to leak out, leading to bacterial cell death due to nutrient deprivation, thereby inhibiting the growth of S. aureus. Peppermint and camphor essential oils have been shown to have anesthetic effects on clownfish keratinocytes and are used in animal laboratory treatment. Clove oil (5 μL / L) and camphor oil (120 μL / L) can sedate fish in water at a density of 5 fish / L for 24 hours.

[0003] An emulsion is a mixed system of two immiscible liquids (usually water and oil) emulsified by an emulsifier, in which the presence of the oil phase allows the dissolution of fat-soluble substances. As a carrier of bioactive substances, it can load and embed some chemically unstable functional factors, greatly expanding its application in the food industry. Low molecular weight emulsifiers (such as Tween and Span) and amphiphilic biopolymers are often used to stabilize heterogeneous emulsions. Emulsifiers prevent droplet aggregation by reducing surface tension through adsorption at the oil / water interface to achieve stability of the emulsion system. Compared with conventional emulsions (micrometer-sized droplets), the small droplet size of nanoemulsions (average particle size of 10 to 100 nm) is believed to provide higher kinetic stability and the ability to resist emulsion instability such as sedimentation or coalescence.

[0004] When the droplet size is between 50 and 200 nm, nanoemulsions usually exhibit transparent or translucent optical properties, which also meets people's sensory needs for some commodities. The biggest difference between nanoemulsions and coarse dispersions such as crude emulsions and suspensions is their stability. These crude dispersions are weakly kinetically stable and thermodynamically unstable systems, and generally phase separation will occur within a few hours to a few days, while nanoemulsions are kinetically stable systems and their stability can be maintained for months or even longer. Generally, the preparation of nanoemulsions requires first forming a coarse emulsion, and then further dispersing it into a nanoemulsion. Nanoemulsions cannot form spontaneously and require energy input and the presence of an emulsifier. Summary of the Invention

[0005] The invention provides preparation of a camphor essential oil nanoemulsion and application of the camphor essential oil as a fish anesthetic in a method for keeping sea bass alive in water. The camphor essential oil nanoemulsion is prepared using Tween 80 as an emulsifier, and properties of the camphor essential oil nanoemulsion, such as particle size, potential, polydispersity index (PDI), biological activity, and stability, are studied. Nanoparticles can solve the problems of poor solubility of essential oil in water and the side effects of ethanol, and the camphor essential oil can be used to achieve anesthesia at a relatively low concentration and promote rapid recovery after anesthesia. The camphor essential oil is added to the transport water and applied to the method for keeping sea bass alive.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a camphor essential oil nanoemulsion specifically comprises the following steps:

[0008] Step S1, Tween 80 is used as an emulsifier. 5 mL of Tween 80 is dissolved in 90 mL of deionized water solution and magnetically stirred (600 rpm) at room temperature for 60 min to prepare a Tween 80 emulsifier.

[0009] Step S2, adding 5 g of camphor essential oil to the magnetically stirred stock solution in S1, and stirring at high speed (10000 rpm) for 10 min to obtain a crude camphor essential oil emulsion with a concentration of 50 mg / mL;

[0010] Step S3, using ultrasonic treatment to treat the crude emulsion, the ultrasonic treatment power density is 573.75W / cm 2 , frequency 20 kHz, time 30 min, and finally the nanoemulsion of camphor essential oil was prepared.

[0011] As a preferred embodiment, the sea bass has a body length of about 35±1.0 cm.

[0012] As a preferred embodiment, the body mass is about 400g±3.5g.

[0013] As a preferred embodiment, the seabreams are temporarily incubated in a 300L food-grade tank in the laboratory for 36h before the experiment begins.

[0014] As a preferred embodiment, the water is replaced every day for 12h in order to maintain a high water quality.

[0015] As a preferred embodiment, the water used in the experiment is prepared from filtered water and sea salt, and is prepared one day before the experiment begins and used after 24h of continuous aeration.

[0016] As a preferred embodiment, the salinity of the seawater is 16-17‰, the dissolved oxygen is 5-7mg / L, and the pH value is 7.5-8.0.

[0017] The application of the cinnamomum camphora nanomulsion prepared by the above method as a fish anesthetic.

[0018] A live-keeping transportation method for seabreams based on a fish anesthetic, comprising the following steps:

[0019] Step (1), the experiment is divided into four groups, which are 20mg / L of cinnamomum camphora emulsified essential oil (NE), 20mg / L of free cinnamomum camphora essential oil (EO), 20mg / L of MS-222 (MS), and a control group (CK) without adding anesthetic and essential oil in the water body;

[0020] Step (2), the seabreams in each group are loaded into a transportation box, the transportation fish-water ratio is 1:5 (the density is 200g / L), the dissolved oxygen (DO) concentration is ensured to be more than 7.0mg / L during the transportation, the water temperature is 20℃, the salinity is 16‰, and the pH value is 7.5;

[0021] Step (3) simulates the transportation of live fish in a vibrating conveyor, and is transported on a B-class road for 1h and then on an A-class road for 5h, the cycle is repeated 4 times, and the transportation lasts for 24h;

[0022] Step (4) is immediately placed in water for recovery for 12h after the transportation.

[0023] Beneficial effects: the cinnamomum camphora nanomulsion can eliminate the problems of poor solubility of essential oil in water and side effects of ethanol, and can have faster anesthetic and recovery times at a lower concentration. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the cinnamomum camphora nanomulsion in embodiment 1 of the present application.

[0025] Figure 2 is a schematic diagram of the ROS content of each experimental group in embodiment 3 of the present application.

[0026] Figure 3Schematic diagram of the changes in catalase (CAT) (A), glutathione peroxidase (GSH-Px) (B), lipid peroxide (LPO) (C) and superoxide dismutase (SOD) (D) in each experimental group in Example 3 of the present invention.

[0027] Figure 4 Schematic diagram of the changes in alanine aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B) and malondialdehyde (MDA) (C) in each experimental group in Example 4 of the present invention.

[0028] Figure 5 Schematic diagram of the pathological changes in gill tissues of each experimental group in Example 4 of the present invention.

[0029] Figure 6 Schematic diagram of the changes in the levels of heat shock protein 70 (HSP70) (A) and heat shock protein 90 (HSP90) (B) in each experimental group in Example 5 of the present invention.

[0030] Figure 7 Schematic diagram of the changes in IL-1B (A), IL-6 (B) and TNF-α (C) levels in each experimental group in Example 5 of the present invention. DETAILED DESCRIPTION

[0031] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0032] Microstructure:

[0033] The physical characteristics of camphor essential oil nanoemulsion were determined by laser particle size analyzer (Zetasizer-Pro).

[0034] Example 1

[0035] Step S1, 5 mL of Tween 80 was taken and dissolved in a deionized water solution (90 mL), and magnetic stirring (600 rpm) was performed at room temperature for 60 min to prepare a Tween 80 emulsifier;

[0036] Step S2: 5 g of camphor essential oil was added to the magnetically stirred stock solution in S1, and the mixture was homogenized at 10,000 rpm for 10 min to obtain a crude camphor essential oil emulsion with a concentration of 50 mg / mL.

[0037] Step S3, treating the crude emulsion with ultrasound at a power density of 573.75 W / cm 2 , frequency 20 kHz, time 30 min, and finally a nanoemulsion of camphor essential oil with a concentration of 20 mg / L was prepared (NE group).

[0038] The sea bass was about 35±1.0 cm in length and about 400 g±3.5 g in weight. Before the experiment, the sea bass was temporarily incubated in a 300 L food-grade tank in the laboratory for 36 h, and the water was changed every 12 h. The experimental water was prepared from filtered water and sea salt, and was prepared one day before the experiment and used after 24 h of continuous aeration. The salinity of the seawater was 16-17‰, the dissolved oxygen was 5-7 mg / L, and the pH value was 7.5-8.0.

[0039] The cinnamomum camphora nanoemulsion was prepared as shown in Figure 1 The prepared emulsion was diluted by an appropriate number of times to prevent the aggregation and fusion of particles, and then was loaded into a polystyrene cuvette for analysis by a laser particle size analyzer. The software parameters were set as follows: dispersant polystyerene latex (RT=1.590, Absorption=0.0010), dispersion phase water, temperature 25°C, weighing interval 120 s, and measurement parallel number automatic. The cuvette was washed with deionized water for 3 times and the emulsion for 3 times, and the particle size, zeta potential, and polydispersity index (PDI) were measured. The average droplet diameter of the cinnamomum camphora nanoemulsion was 61.27±0.34 nm, which met the standard of nanoemulsion. The PDI was an index of multiple scattering and also an index for measuring the uniformity and stability of droplet size. The PDI index of the cinnamomum camphora nanoemulsion was 0.277±0.005, indicating that the system was uniformly distributed. The zeta potential value of the cinnamomum camphora nanoemulsion was 13.47 mV, and the system was stable, which was due to the Tween 80 surfactant ensuring the stability of the prepared emulsion of essential oil.

[0040] Example 2

[0041] Referring to Example 1, 20 mg / L of free cinnamomum camphora essential oil (EO group), 20 mg / L of MS-222 (MS group), and no anesthetic addition group (CK group) were used as controls.

[0042] Fresh and live sea bass (body weight 400 g±3.5 g) was purchased from a Shanghai aquatic product market, and was transported at a fish-to-water ratio of 1:5 by 20 mg / L of cinnamomum camphora nanoemulsion, 20 mg / L of free cinnamomum camphora essential oil, 20 mg / L of MS-222 anesthesia, and a no-anesthesia group. The transportation of live fish in a vibrating conveyor was simulated, and the fish was transported on a B-class road for 1 h and then on an A-class road for 5 h, for a total of 4 cycles. After transportation, the fish was immediately put into water for recovery for 12 h. The cinnamomum camphora nanoemulsion in this example was used to determine the anesthesia time for 24 h of transportation.

[0043] The survival rate of 24h transportation with these three anesthetics was 100%. The experimental results showed that the cinnamomum camphora nanoemulsion greatly shortened the sedation time and was lower than that of MS-222. The sedative activity after the nanoemulsification process was more effective because the emulsion could increase the hydrophilicity of the essential oil and produce a protective effect against the side effects of the essential oil. Similar to the absorption process, the elimination of drugs or exogenous substances is carried out through the gills, and depends on the species of fish and the physicochemical characteristics of the compound, etc. The nanoemulsion can increase the contact area with the fish, and under the condition of the same essential oil concentration, the cinnamomum camphora nanoemulsion can more effectively and safely exert the sedative effect of the essential oil.

[0044] Table 1 Comparison of sedation time of 20mg / L anesthetics

[0045]

[0046] Example 3

[0047] Referring to Example 1, the control group was 20mg / L free cinnamomum camphora essential oil, 20mg / L MS-222 and no anesthetic addition group.

[0048] Freshly caught Epinephelus awoara (body weight 400g±3.5g) were purchased from Shanghai aquatic product market, and were transported according to a fish to water ratio of 1:5 by 20mg / L cinnamomum camphora nanoemulsion, 20mg / L free cinnamomum camphora essential oil, 20mg / L MS-222 anesthesia, and a no anesthetic group was set up. The transportation of live fish in a vibrating conveyor was simulated, and the fish were transported on a B-class road for 1h and then on an A-class road for 5h, for a total of 4 cycles. The fish were immediately placed in water for recovery for 12h after transportation. The cinnamomum camphora nanoemulsion in this example was used to determine the survival rate after 24h transportation and oxidative stress.

[0049] The survival rate of 24h transportation with these three anesthetics was 100%. The experimental results showed that the cinnamomum camphora nanoemulsion greatly shortened the sedation time and was lower than that of MS-222. The sedative activity after the nanoemulsification process was more effective because the emulsion could increase the hydrophilicity of the essential oil and produce a protective effect against the side effects of the essential oil. Similar to the absorption process, the elimination of drugs or exogenous substances is carried out through the gills, and depends on the species of fish and the physicochemical characteristics of the compound, etc. The nanoemulsion can increase the contact area with the fish, and under the condition of the same essential oil concentration, the cinnamomum camphora nanoemulsion can more effectively and safely exert the sedative effect of the essential oil, and can alleviate the oxidation of the fish during transportation. The cinnamomum camphora nanoemulsion in this example was used to determine the tissue damage after 24h transportation.

[0050] Transport-induced damage is associated with excessive ROS production. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) are important protective enzymes that play a crucial role in eliminating free radicals. Antioxidant enzyme activity increased in the control group with increasing transport time: SOD activity peaked at 12 hours, CAT activity peaked at 24 hours after transport, and GSH-Px activity peaked at 24 hours after transport. After 12 hours of recovery, the activities of all three began to decline. Treatment with free Cinnamomum camphora essential oil and Cinnamomum camphora nanoemulsion did not induce a significant response in the first line of defense against oxidative toxicity. Lipid peroxide (LPO) clearance is a rapid response to hyperoxidative conditions, occurring through the regulation of both enzymatic and non-enzymatic antioxidant defense systems. Compared with the control and MS groups, serum GSH-Px activity increased and LPO accumulation decreased in the experimental groups supplemented with essential oil (NE and EO groups), indicating that essential oils can mitigate oxidative damage caused by transport stress. Results for ROS levels also confirmed this finding. At the same time, we found that although the antioxidant enzyme activity of the NE group remained basically unchanged, the ROS and LPO levels were significantly lower than those of the other experimental groups (P<0.05). This indicates that the NE group had a significant sedative effect and produced relatively small oxidative reactions. We found that in the NE group. In contrast, in the MS group, even though the transport stress enhanced the activity of antioxidant enzymes (SOD and CAT) and activated the antioxidant system 24 hours after transportation, it did not completely eliminate ROS (such as Figure 2 As shown), the oxidative stress product LPO content increased (as shown Figure 3 shown).

[0051] Example 4

[0052] Referring to Example 1, the control groups were 20 mg / L free camphor essential oil, 20 mg / L MS-222, and no anesthetic added.

[0053] Fresh sea bass (weighing 400g ± 3.5g) were purchased from the Shanghai Aquatic Products Market and anesthetized with 20mg / L camphor nanoemulsion, 20mg / L free camphor essential oil, and 20mg / LMS-222, and a non-anesthetized group was set up and transported at a fish-water ratio of 1:5. The transport of live fish in a vibrating conveyor was simulated, and the fish were transported on a Class B road for 1 hour and then on a Class A road for 5 hours, for a total of 4 cycles. Immediately after transportation, the fish were placed in water to recover for 12 hours. The camphor nanoemulsion in this example was used to measure tissue damage after 24 hours of transportation.

[0054] Reactive oxygen species induce oxidative stress and liver cell damage, which leads to the leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) into the circulation, resulting in elevated levels of liver-specific and sensitive enzymes ALT and AST in the blood. According to current results, transport is stressful and produces excessive ROS that damage liver cells. Figure 4 Using camphor essential oil and nanoemulsion to calm sea bass before transportation has a hepatoprotective and / or anti-stress effect on the fish. In addition, compared with the CK group, the gill tissue showed epithelial edema and epithelial lifting after 24 hours of transportation. The MS group even showed problems such as lamina hyperplasia and increased chloride cells. However, the NE and EO groups only showed a small amount of epithelial edema and epithelial lifting. Figure 5 This shows that camphor essential oil can effectively protect tissues.

[0055] Example 5

[0056] Referring to Example 1, the control groups were 20 mg / L free camphor essential oil, 20 mg / L MS-222, and no anesthetic added.

[0057] Fresh sea bass (weighing 400g ± 3.5g) were purchased from the Shanghai Aquatic Products Market and anesthetized with 20mg / L camphor nanoemulsion, 20mg / L free camphor essential oil, and 20mg / L MS-222. A non-anesthetized group was also established and transported at a 1:5 fish-to-water ratio. To simulate the transport of live fish on a vibrating conveyor, the fish were transported on a Class B road for 1 hour, then on a Class A road for 5 hours, for a total of 4 cycles, for a total of 24 hours. After transport, the fish were immediately placed in water for 12 hours of recovery. In this example, the camphor nanoemulsion was used to measure immune responses.

[0058] Serum lysozyme (LZM) is an important enzyme with bactericidal activity and an indicator of the innate immune system in fish. Enzyme activity increases during stress and infection to enhance host defense; however, decreased enzyme activity is an indicator of deteriorating health. After 24 hours of transport, LZM activity in the CK group increased with increasing transport time, but LZM activity in the NE group remained relatively stable throughout the entire process. This is attributed to the sedative effect of the nanoemulsion, which alleviated the stress response caused by transport. Serum lysozyme is an important nonspecific immune factor in fish and a commonly used parameter for evaluating fish immune function under stressful conditions.

[0059] Cells often respond to stress through changes in gene expression, such as upregulation of the expression levels of highly conserved proteins collectively known as heat shock proteins (HSPs). Figure 6 As shown, 24 hours after transportation, the HSP70 level in the NE group was lower than that in the other experimental groups. This is because the sedative effect of NE was better than that in the other experimental groups, resulting in a smaller stress response.

[0060] Cytokines (IL-1β, IL-6, TNF-α) are key proinflammatory factors that lead to the activation of inflammatory responses. They are the most commonly used immune marker genes in fish because toxic substances, pathogenic microorganisms and stress can upregulate their expression levels. Among them, TNF-α is an important component of the TNF family, which can cause inflammation and stimulate the immune system. Activated TNF-α can activate a variety of proinflammatory factors, including IL-6 and IL-1β. Transport-induced stress leads to upregulation of IL-1β, IL-8 and TNF-α expression levels in the head kidney. In this experiment, the inflammatory factors TNF-α, IL-6 and IL-1β in each experimental group were upregulated and returned to the initial levels after 12 hours of recovery.

[0061] like Figure 7 As shown in the results, the changes in inflammatory factors in the essential oil and NE groups were relatively stable throughout the process and were significantly lower than those in the MS and CK groups, indicating that camphor essential oil can effectively alleviate the inflammatory response caused by transportation stress. At the same time, we found that the changes in inflammatory factors in the essential oil and NE groups were relatively stable throughout the process and were significantly lower than those in the MS and CK groups, indicating that camphor essential oil can effectively alleviate the inflammatory response caused by transportation stress.

[0062] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a camphor essential oil nanoemulsion, characterized in that: The specific steps include: Step S1, Tween 80 is used as an emulsifier, 5 mL of Tween 80 is dissolved in deionized water, and magnetic stirring is performed at room temperature for 60 minutes to prepare a Tween 80 emulsifier; Step S2, adding 5 g of camphor essential oil to the magnetically stirred stock solution in S1, and homogenizing and stirring at high speed for 10 min to obtain a crude camphor essential oil emulsion with a concentration of 50 mg / mL; Step S3, using ultrasonic treatment to treat the crude emulsion, the ultrasonic treatment power density is 573.75W / cm 2 , frequency 20 kHz, time 30 min, and finally the nanoemulsion of camphor essential oil was prepared.

2. The method for preparing a camphor essential oil nanoemulsion according to claim 1, wherein The body length of sea bass is about 35±1.0cm.

3. The method for preparing a camphor essential oil nanoemulsion according to claim 1, wherein The body mass is approximately 400g±3.5g.

4. The method for preparing a camphor essential oil nanoemulsion according to claim 1, wherein Before the start of the experiment, sea bass were temporarily incubated in 300 L food-grade tanks in the laboratory for 36 h.

5. The method for preparing a camphor essential oil nanoemulsion according to claim 1, wherein In order to maintain high water quality, the water was changed every 12 hours every day.

6. The method for preparing a camphor essential oil nanoemulsion according to claim 1, wherein The experimental water was prepared from filtered water and sea salt, prepared one day before the experiment and used after continuous aeration for 24 h.

7. The method for preparing a camphor essential oil nanoemulsion according to claim 1, wherein The salinity of seawater is 16-17‰, the dissolved oxygen is 5-7 mg / L, and the pH value is 7.5-8.

0.

8. Use of the camphor essential oil nanoemulsion as claimed in claim 1 as a fish anesthetic.

9. A method for transporting sea bass alive based on fish anesthetics, characterized in that: The steps include: Step (1), the experiment was divided into four groups, namely 20 mg / L of emulsified camphor essential oil, 20 mg / L of free camphor essential oil, 20 mg / L of MS-222, and a control group in which no anesthetic and essential oil were added to the water; Step (2), the sea bass of each group are loaded into a transport box, the fish-to-water ratio is 1:5, and during transportation, the dissolved oxygen concentration is ensured to be greater than 7.0 mg / L, the water temperature is 20°C, the salinity is 16‰, and the pH is 7.5; Step (3) simulates the transportation of live fish in a vibrating conveyor, transporting them on a Class B road for 1 hour, then transporting them on a Class A road for 5 hours, and repeating this cycle 4 times for a total of 24 hours; Step (4) After transport, immediately place in water to recover for 12 hours.

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