Preparation containing tyramine and use thereof

Abstract

wt% of polyethylene glycol (PEG). Accordingly, the duration of action of tyramine can be extended, the application frequency of tyramine can be decreased, and thus, the cost in aquaculture can be reduced. The use of the preparation containing tyramine is also disclosed.

Description

Tyramine preparations and their uses This invention relates to a tyramine formulation, particularly a long-acting tyramine formulation. The invention also relates to the use of this tyramine formulation. In recent years, shrimp farming in Taiwan has flourished. Shrimp not only serve as a food source but also generate substantial economic benefits for the country through exports. Due to the rapid development of shrimp farming, coupled with high land prices, farmers are forced to adopt high-density farming methods to reduce costs. High-density farming can lead to a deterioration of the farming environment, making it easier for shrimp to spread diseases and thus increasing the mortality rate. In order to reduce the mortality rate, farmers often add antibiotics to the feed. However, the overuse of antibiotics not only causes allergies in consumers who eat shrimp, but may also induce antibiotic resistance in pathogens. Therefore, further improvements are still needed. For example, tyramine is a biogenic monoamine known to be used to enhance the performance of whiteleg shrimp (Scaevola equina). Litopenaeus vannamei and freshwater long-armed prawns ( The immunity of shrimp species such as Macrobrachium rosenbergii can enhance their resistance to pathogens (e.g., "Tyramine's modulation of immune resistance functions in..."). Litopenaeus vannamei and its signal pathway" and "The temporary modulation of tyramine on immune responses, carbohydrate metabolism, and catecholamines in As revealed in journal articles such as "Macrobrachium rosenbergii," tyramine's effect on shrimp is short-lived (e.g., only about 96 hours in whiteleg shrimp). Therefore, to ensure its sustained effect, aquaculture farmers need to administer tyramine multiple times, increasing farming costs. In light of this, there is indeed a need for a long-acting tyramine formulation and its applications. To address the above problems, the purpose of this invention is to provide a tyramine formulation with a longer duration of action. A secondary objective of this invention is to provide a use of tyramine preparations that can enhance the immunity of shrimp, thereby eliminating the need for antibiotic users. The use of the quantifiers "a" or "an" for the elements and components described throughout this invention is for convenience and to provide the general meaning of the scope of the invention; in this invention, it should be interpreted as including one or at least one, and the concept of a single also includes the case of a plural, unless it clearly means otherwise. The tyramine formulation of the present invention may comprise: 12.5 to 25.0% tyramine and 75.0 to 87.5% polyethylene glycol by weight, wherein the molecular weight of the polyethylene glycol is between 950 and 1,050, and the average molecular weight of the polyethylene glycol is 1,000. For example, the weight ratio of tyramine to polyethylene glycol may be 1:5. Accordingly, the tyramine formulation of the present invention, through the combination of tyramine and polyethylene glycol, can prolong the duration of tyramine's effect in promoting the phagocytic activity of shrimp hemocytocytes, thereby reducing shrimp mortality caused by pathogen attacks. By selecting polyethylene glycol with a specific molecular weight, the combined effect of tyramine and polyethylene glycol can be enhanced. In the tyramine formulation of the present invention, tyramine and polyethylene glycol are mixed at a temperature of 40°C to form the tyramine formulation. This enhances the synergistic effect of tyramine and polyethylene glycol. The use of the tyramine preparation of the present invention is to prepare a drug that enhances the immune capacity of shrimp. The tyramine preparation is administered to a shrimp (e.g., whiteleg shrimp) to enhance the shrimp's immune capacity, and the tyramine preparation is as described above. Accordingly, the use of the tyramine preparation of the present invention, by combining tyramine with polyethylene glycol, can prolong the duration of tyramine's effect in promoting the phagocytic activity of shrimp hemoglobin cells, thereby reducing the frequency of tyramine administration and achieving the effect of reducing the cost of aquaculture. The tyramine preparation of the present invention can be injected into the shrimp, preferably into the cephalothorax ventral sinus of the shrimp; in this way, the tyramine preparation can flow to various tissues through the hemolymph of the shrimp, thereby having a better effect on enhancing the shrimp's immunity. The use of the tyramine preparation of the present invention is wherein the tyramine preparation is injected into the shrimp at a dose of 50-100 ng per shrimp; thus, the dosage of the tyramine preparation is used to enhance the shrimp's immune capacity. To make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments of the present invention are described below in detail with reference to the accompanying drawings. The term "shrimp" as used in this invention can refer to farmed shrimp, including but not limited to, whiteleg shrimp (Scaevola valens). Litopenaeus vannamei), grass shrimp ( Penaeus monodon, tiger prawn ( Marsupenaeus japonicus, Chinese grass shrimp ( Fenneropenaeus chinensis, Indian shrimp ( Fenneropenaeus indicus), sand shrimp ( Metapenaeus ensis barbata, red-tailed shrimp ( Penaeus penicillatus and freshwater long-armed prawns ( Macrobrachium rosenbergii). The tyramine formulation of one embodiment of the present invention may contain tyramine and polyethylene glycol, for example, it may contain 12.5 to 25.0% tyramine and 75.0 to 87.5% polyethylene glycol by weight percentage. Specifically, in this embodiment, polyethylene glycol with a molecular weight between 950 and 1,050 and an average molecular weight of 1,000 is selected. After weighing an appropriate amount of tyramine and polyethylene glycol, the tyramine and polyethylene glycol are melted and mixed at a temperature of 40°C to obtain the tyramine formulation. This tyramine preparation can increase the total number of hemoglobin cells in shrimp, thereby enhancing the phagocytic activity of hemoglobin cells and reducing the mortality caused by pathogen attacks on shrimp. Therefore, an effective dose of tyramine preparation can be administered to the shrimp to enhance its immune capacity. For example, the tyramine preparation can be administered to the shrimp via injection. For instance, the tyramine preparation can be injected into the ventral sinusoids of the shrimp's cephalothorax. These ventral sinusoids are close to the heart and are a concentration point of hemolymph in the shrimp, allowing the tyramine preparation to flow to various tissues via the shrimp's hemolymph. In this embodiment, shrimp weighing approximately 8.5 ± 0.1 grams are selected, and the tyramine preparation is administered at a dose of 5.81–11.91 ng per gram of shrimp body weight, resulting in approximately 50–100 ng of tyramine preparation per shrimp. To verify that the tyramine preparation of this invention does indeed have the activity of enhancing the immune capacity of shrimp, whiteleg shrimp obtained from the Pingtung University of Science and Technology farm were selected. These whiteleg shrimp were first cultured in indoor concrete tanks for two weeks to allow them to adapt to the experimental culture environment (salinity 20‰; water temperature 28±1℃; pH 7.5). Then, the following experiments were conducted: (A) Effects on immune parameters Please refer to Table 1. In this experiment, tyramine (0.686 mg / L, dissolved in physiological saline, 20 μL / shrimp body) was injected into the whiteleg shrimp in group A1, polyethylene glycol (3.43 mg / L, dissolved in physiological saline, 20 μL / shrimp body) was injected into the whiteleg shrimp in group A2, and the aforementioned tyramine preparation (containing 0.686 mg / L tyramine and 3.43 mg / L polyethylene glycol, dissolved in physiological saline, 20 μL / shrimp body) was injected into the whiteleg shrimp in group A3. The whiteleg shrimp in group A0, which were injected with physiological saline, served as the control group. Next, at the time of the aforementioned treatment, and at time points of 0.5, 1, 2, 4, 8, and 16 hours after the treatment, hemolymph was extracted from the whiteleg shrimp, and the total hemocyte count (THC) in the hemolymph of each group of whiteleg shrimp was calculated. The higher the total hemocyte count, the stronger the immune system. Table 1. Treatment conditions of whiteleg shrimp in each group of this experiment. Referring to Figure 1, in Group A1, an increase in total red blood cell count was observed in the white shrimp after injection of tyramine starting at 0.5 hours, and this increase continued until the 2nd hour. However, starting at the 4th hour after injection, the total red blood cell count in Group A1 returned to normal (no difference from Group A0). Conversely, in Group A3, an increase in total red blood cell count was also observed starting at 0.5 hours after injection of the tyramine preparation. Although the effect was no different from that in Group A1, the duration of the effect of the tyramine preparation was longer, lasting until the 8th hour, indicating that the polyethylene glycol compound can indeed prolong the duration of tyramine's effect. (B) Efficacy against Vibrio alginolyticus Please refer to Table 2. In this experiment, tyramine (0.686 mg / L, 20 μL / shrimp body), polyethylene glycol (3.43 mg / L, 20 μL / shrimp body), and the aforementioned tyramine preparation (containing 0.686 mg / L tyramine and 3.43 mg / L polyethylene glycol, 20 μL / shrimp body) were injected into groups B1, B2, and B3 of whiteleg shrimp, respectively. Group B0, which was injected with physiological saline, served as the control group. Next, at 0, 0.5, 1, 2, 4, 8, and 16 hours after the aforementioned treatment, Vibrio alginolyticus was injected into each group of whiteleg shrimp to infect them with Vibrio alginolyticus. Vibro algonilyticus, infectious dose 2×10 7 (CFU / shrimp body), then hemolymph was extracted, and the phagocytic activity of hemolymph cells in the hemolymph of each group of white shrimp was further tested to evaluate the efficacy against Vibrio alginolyticus. Table 2. Treatment conditions of whiteleg shrimp in each group of this experiment. Please refer to Figure 2. In Group B1, increased phagocytic activity of hemoglobin cells was observed in the shrimp after injection of tyramine starting from the first hour, continuing until the fourth hour. However, from the eighth hour after injection, the phagocytic activity of hemoglobin cells in Group B1 returned to normal (no difference from Group B0). Conversely, in Group B3, increased phagocytic activity of hemoglobin cells was also observed starting from the first hour after injection of the tyramine preparation. Although the effect was similar to that of Group B1, the duration of the effect of this tyramine preparation was longer, lasting until the 16th hour. This indicates that the combination with polyethylene glycol can also prolong the duration of tyramine's efficacy in clearing pathogens. (C) Survival rate after Vibrio alginolyticus infection Next, referring to Table 3, this experiment also administered tyramine (0.686 mg / L, 20 μL / shrimp body), polyethylene glycol (3.43 mg / L, 20 μL / shrimp body), and the aforementioned tyramine preparation (containing 0.686 mg / L tyramine and 3.43 mg / L polyethylene glycol, 20 μL / shrimp body) to the whiteleg shrimp in groups C2, C3, and C4, respectively. The whiteleg shrimp in groups C0 and C1, which were injected with physiological saline, served as the control group. Next, 16 hours after the aforementioned treatment, Vibrio alginolyticus was injected into groups C1–C4 of whiteleg shrimp to infect them (infection dose of 2 × 10⁻⁶). 5 (CFU / shrimp body), and the mortality rate of each group of whiteleg shrimp was calculated at 0, 24, 48, 72, 96, 120, 168 and 168 hours. Table 3. Treatment conditions of whiteleg shrimp in each group of this experiment. Please refer to Figure 3. Although the mortality rate of whiteleg shrimp in group C2 was no different from that in group C1, a significant decrease in mortality rate was observed in group C4 starting from the 120th hour. This indicates that the polyethylene glycol compound can also prolong the duration of tyramine's efficacy in eliminating pathogens, thereby reducing the risk of whiteleg shrimp dying from Vibrio alginolyticus infection. In summary, the tyramine preparation and its uses of the present invention, through the combination of tyramine and polyethylene glycol, can prolong the duration of tyramine's effect in promoting the phagocytic activity of shrimp hemocytocytes, thereby reducing the mortality of shrimp caused by pathogen attacks. Thus, the frequency of tyramine administration can be reduced, achieving the effect of reducing the cost of aquaculture. Although the present invention has been disclosed using the above preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art to the above embodiments without departing from the spirit and scope of the present invention shall still fall within the scope of the technology protected by the present invention. Therefore, the scope of protection of the present invention shall include all changes within the meaning and equivalent scope of the appended claims. none [Figure 1] Bar chart showing the total red blood cell count of whiteleg shrimp in groups A0-A3 at different time points after treatment in Experiment (A). If the letters indicating the same time point on the bar chart differ, it indicates a significant difference in the total red blood cell count. p < 0.05, n = 6). [Figure 2] Bar graph showing the phagocytic activity of hemoglobin cells at different time points after treatment in groups B0-B3 of whiteleg shrimp in experiment (B). Different letters indicating the same time point on the bar graph indicate a significant difference in phagocytic activity of the hemoglobin cells. p < 0.05, n = 6). [Figure 3] Line graph showing the survival rate of whiteleg shrimp in groups C0-C4 within 168 hours after treatment in experiment (C). Different letters indicating the same time point on the line graph indicate a significant difference in survival rate. p < 0.05, n = 3).

Claims

1. A tyramine formulation comprising: 12.5 to 25.0% tyramine and 75.0 to 87.5% polyethylene glycol by weight, wherein the molecular weight of the polyethylene glycol is between 950 and 1,050, and the average molecular weight of the polyethylene glycol is 1,000. For example, the tyramine formulation in claim 1, wherein... The weight ratio of tyramine to polyethylene glycol is 1:

5. For example, the tyramine preparation of any one of claims 1 to 2, wherein, Tyramine and polyethylene glycol are mixed at 40°C to form the tyramine formulation. The use of a tyramine preparation is for preparing a drug that enhances the immune capacity of shrimp. The tyramine preparation is administered to a shrimp to enhance its immune capacity, and the tyramine preparation is any one of claims 1 to 3. For example, the use of tyramine preparations as requested in claim 4, wherein, The tyramine preparation was injected into the shrimp. For example, the use of tyramine preparations as requested in claim 5, wherein, The tyramine preparation was injected into the ventral sinusoids of the cephalothorax of the shrimp. For example, the use of tyramine preparations as described in any of claims 4 to 6, wherein, The tyramine preparation is administered to the shrimp by injection at a dose of 50-100 ng per shrimp. For example, the use of tyramine preparations as described in any of claims 4 to 6, wherein, The shrimp is a whiteleg shrimp.

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