Application of paeonol in preparation of shrimp enterocytozoon hepatopenaei inhibitor

Abstract

The invention discloses an application of paeonol in the aspect of preparing a shrimp enterocytozoon hepatopenaei inhibitor, discloses an application of paeonol in the aspect of preparing a medicine for preventing and / or treating shrimp enterocytozoon hepatopenaei disease, and discloses a paeonol preparation for resisting shrimp enterocytozoon hepatopenaei and a preparation method of the paeonol preparation. The composition is characterized by being prepared from the following raw materials in percentage by mass: 5-13% of paeonol, 2-5% of ethanol, 2-4% of tween-20, 1-3% of water-soluble azone and the balance of peanut oil. The preparation method comprises the following steps: weighing the paeonol, the tween-20, the water-soluble azone, the ethanol and the peanut oil for later use; the preparation method comprises the following steps: uniformly mixing paeonol, ethanol and peanut oil until the paeonol is completely dissolved, then adding tween-20 and water-soluble azone, and uniformly mixing to obtain the paeonol preparation. The paeonol preparation has the advantages of inhibiting the proliferation of enterocytozoon hepatopenaei in shrimp bodies, improving the weight gain rate of prawns, effectively reducing the economic loss caused by the outbreak of the enterocytozoon hepatopenaei to prawn culture, and being low in compound cost, simple in preparation process and suitable for industrial production. The use method is simple.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to the application of paeonol in the preparation of an inhibitor against Enterocytozoon hepatopenaei. Background Art

[0002] Enterocytozoon hepatopenaei ( Enterocytozoon hepatopenaei , abbreviated as EHP) is an obligate intracellular parasite that seriously threatens the sustainable development of aquaculture. EHP can infect Litopenaeus vannamei ( Litopenaeus vannamei ), Penaeus monodon ( Penaeus monodon ), Macrobrachium rosenbergii ( Macrobrachium rosenbergii ), Procambarus clarkii ( Procambarus clarkii ), etc., causing a decline in the feeding ability of shrimp, slow or even stagnant growth, leading to wasted feed and prone to secondary bacterial diseases at the same time. It has the characteristics of multiple infection vectors, wide distribution, and strong transmission ability, seriously threatening industrial safety. As an important part of new fishery drugs, Chinese herbal medicines have the advantages of low toxicity, low residue, and high biological safety. Feed additives such as their powders or crude extracts can improve the immunity of aquatic animals and increase the resistance to pathogen infection; moreover, the discovery of natural active monomers from them can more precisely target pathogens and reduce or avoid the "drug hazards" brought about by the current use of agricultural and veterinary drugs, and has become one of the research hotspots of safe and green fishery prevention and treatment drugs today.

[0003] Paeonol is derived from the dried root bark of the Ranunculaceae plant Paeonia suffruticosa, and its structural formula is: .

[0004] Paeonol is also known as paeonol, with a molecular formula of C9H 10 O3, a molecular weight of 166.17, soluble in methanol or ethanol, soluble in hot water, and insoluble in cold water. There is currently no research and application in anti-EHP. Summary of the Invention

[0005] One of the technical problems to be solved by the present invention is to provide an application of paeonol in the preparation of an inhibitor against Enterocytozoon hepatopenaei; the second is to provide an application of paeonol in the preparation of a drug for preventing and / or treating Enterocytozoon hepatopenaei disease, and the third is to provide a paeonol preparation for inhibiting Enterocytozoon hepatopenaei and its preparation method, and this preparation can effectively inhibit the replication of EHP in shrimp.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: The present invention provides an application of paeonol in the preparation of an inhibitor against Enterocytozoon hepatopenaei.

[0007] The present invention also provides an application of paeonol in the preparation of a drug for preventing and / or treating Enterocytozoon hepatopenaei disease.

[0008] The present invention also provides a paeonol preparation for resisting Enterocytozoon hepatopenaei, which is composed of the following raw materials and their mass percentages: paeonol 5-13%, ethanol 3-5%, Tween-20 2-4%, water-soluble azone 1-3%, and the rest is peanut oil.

[0009] Preferably, the paeonol preparation is composed of the following raw materials and their mass percentages: paeonol 13.0%, ethanol 5.0%, peanut oil 75.0%, Tween-20 4.0%, and water-soluble azone 3.0%.

[0010] Preferably, the paeonol preparation is composed of the following raw materials and their mass percentages: paeonol 13.0%, ethanol 5.0%, peanut oil 77.0%, Tween-20 3.0%, and water-soluble azone 2.0%.

[0011] Preferably, the paeonol preparation is composed of the following raw materials and their mass percentages: paeonol 13.0%, ethanol 5.0%, peanut oil 79.0%, Tween-20 2.0%, and water-soluble azone 1.0%.

[0012] The present invention also provides a preparation method of the paeonol preparation for inhibiting Enterocytozoon hepatopenaei, including the following steps: weighing paeonol, Tween-20, water-soluble azone, ethanol, and peanut oil for standby; mixing paeonol, ethanol, and peanut oil until the paeonol is completely dissolved, and then adding Tween-20 and water-soluble azone and mixing evenly to obtain the paeonol preparation.

[0013] The present invention also provides the application of the above-mentioned paeonol preparation for inhibiting Enterocytozoon hepatopenaei in penaeid shrimp farming, and the addition concentration of the paeonol preparation in the penaeid shrimp farming water is 30 mg / L.

[0014] Compared with the prior art, the advantages of the present invention are as follows: the present invention discloses for the first time the application of paeonol in the preparation of inhibitors against Enterocytozoon hepatopenaei and drugs for preventing or treating Enterocytozoon hepatopenaei disease. As the active ingredient of a new type of medicament, paeonol has low toxicity and is not prone to drug resistance, and can effectively prevent and treat Enterocytozoon hepatopenaei disease. Furthermore, the paeonol preparation is disclosed. As a new type of fishery drug, the paeonol preparation is natural, pollution-free, not prone to drug resistance in penaeid shrimp, and can effectively prevent and treat Enterocytozoon hepatopenaei disease. The preparation has low cost and simple usage method, which is conducive to popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the analysis results of the toxicity and anti-EHP activity of paeonol, wherein A is the survival rate of healthy shrimps under different concentrations of paeonol, and B is the EHP copy number in diseased shrimps under different concentrations of paeonol; Figure 2For the preventive effect of paeonol on EHP, where A is the measurement result of the EHP copy number after healthy shrimps were pretreated with paeonol and then infected with EHP, and B is the measurement result of the body weight of healthy shrimps after being pretreated with paeonol and then infected with EHP; Figure 3 For the therapeutic effect of paeonol on EHP, where A is the measurement result of the EHP copy number after healthy shrimps were infected with EHP and then treated with paeonol, and B is the measurement result of the body weight of healthy shrimps after being infected with EHP and then treated with paeonol; Figure 4 For the influence result of paeonol on the horizontal transmission of EHP when healthy shrimps and diseased shrimps cohabited, where * indicates p < 0.05 and ** indicates p < 0.01. Detailed implementation manners

[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0017] To clarify the essence of the present invention, Litopenaeus vannamei was used as the experimental object to conduct the detection of the anti-EHP activity of paeonol and the pharmacological experiment of its preparation to prove its use in anti-EHP.

[0018] Specific Example 1. Analysis of the anti-EHP activity of paeonol.

[0019] 1. Experimental materials: Paeonol, dimethyl sulfoxide (DMSO), and absolute ethanol were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. EHP and Litopenaeus vannamei were provided by the Zhejiang Ocean Fisheries Research Institute. Preparation of the paeonol stock solution: Weigh paeonol and fully dissolve it with DMSO to prepare a stock solution with a final concentration of 50000 mg / L and store it at 4°C for later use.

[0020] 2. Detection of EHP copy number: Use a rapid extraction kit for genomic DNA of marine animals (Tiangen) to extract the DNA of the hepatopancreas of shrimps. After detecting the DNA concentration (adjust the final concentration to 100 ng / μL) and purity with a ultra-micro spectrophotometer, perform qPCR amplification experiments. The qPCR amplification primers include ENF 185 Forward and reverse amplification primers, ENF 185 The nucleotide sequence of the forward amplification primer is shown as SEQ ID NO.1: 5'-GTAGCGGAACGGATAGGG-3', ENF 185The nucleotide sequence of the reverse amplification primer is shown as SEQ ID NO.2: 5'-CCAGCATTGTCGGCATAG-3'. The qPCR amplification reaction system is as follows: 7.5 μL of 2 × Es Taq Master Mix, 0.3 μL of 10 μM forward amplification primer, 0.3 μL of 10 μM reverse amplification primer, 1 μL of template DNA, and 5.9 μL of ddH2O. The qPCR amplification reaction program is as follows: 95°C for 5 min; 95°C for 30 s, 55.5°C for 30 s, 72°C for 30 s, for 35 cycles; 72°C for 10 min. According to the standard curve made from the pMD19T-ENF 185 standard product, the qPCR results are converted to obtain the EHP copy number.

[0021] 3. Paeonol toxicity experiment: Take healthy prawns and divide them into 5 groups, with 15 prawns in each group. By means of soaking, the experiment is set with the concentrations of paeonol in the culture water being 5, 10, 20, 40, and 80 mg / L respectively, and the control group is with the mass concentration of DMSO in the culture water being 0.02%. The survival rate of prawns is counted every 24 h, and the safe concentration of paeonol is determined according to the survival rate of prawns.

[0022] The results are as Figure 1 shown in A below. Compared with the control group, when the concentration of paeonol is 20, 40, and 80 mg / L, the survival rate of prawns decreases significantly; while when the concentration is 5 and 10 mg / L, there is no significant difference. In summary, a concentration of 10 mg / L is selected for subsequent experiments.

[0023] 4. Detection of the anti-EHP activity of paeonol: The experiment is set with five groups of culture water added with paeonol concentrations of 0 mg / L, 1.25 mg / L, 2.5 mg / L, 5 mg / L, and 10 mg / L respectively. Each group is fed with the fresh hepatopancreas of prawns infected with EHP (the EHP concentration in the fresh hepatopancreas of prawns infected with EHP is 1.6×10 6 copies / mg tissue, the same for the following experiments), and the feeding amount is 2 g / 10 tails. The control group is to add the fresh hepatopancreas of prawns infected with EHP in the culture water containing 0.02 wt% DMSO, and the feeding amount is 2 g / 10 tails. Each group has 10 prawns. On the 7th day after infection, samples are taken and the EHP copy number is detected by qPCR.

[0024] The results are as Figure 1As shown in B, there is a correlation between the concentration of paeonol and the EHP copy number. Compared with the control group, when the treatment concentrations of paeonol were 5 and 10 mg / L, the EHP copy number in shrimp significantly decreased, and the inhibition rates were 89% and 48% respectively; while at 1.25 and 2.5 mg / L, the inhibition rates on the EHP copy number were 27% and 16% respectively, but the differences were not significant. These results indicate that paeonol can significantly inhibit the proliferation of EHP at 10 mg / L.

[0025] 5. Preventive effect of paeonol on EHP: Healthy shrimp were divided into two groups, with 30 shrimp in each group. The culture water of the experimental group contained 10 mg / L of paeonol, and the shrimp were fed three times a day. The culture water containing paeonol was changed every 3 days, denoted as EHP 丹皮酚 . The culture water of the control group contained 0.02% DMSO, denoted as EHP DMSO , and the shrimp were fed three times a day. The culture water containing DMSO was changed every 3 days. After 15 d, both groups of shrimp were fed with fresh hepatopancreas of EHP-infected shrimp at a feeding amount of 2 g / 10 shrimp, and qPCR was used to detect the EHP copy number at 15, 30, and 45 d after EHP infection. At the same time, the two groups of shrimp were weighed at 30 d, and the results are as Figure 2 shown.

[0026] As Figure 2 shown in A, after the shrimp were pretreated with paeonol and then infected with EHP, at 15 and 30 d after infection, the EHP copy number in the experimental group of shrimp was significantly lower than that in the control group, decreasing by 66% and 41% respectively; at 45 d after infection, the EHP copy number in the experimental group of shrimp decreased by 25% compared with the control group, but the difference was not significant. As Figure 2 shown in B, at 15, 30, and 45 d after infection, it was found that the weight of the experimental group of shrimp increased compared with the control group, and the weight gain rates were 19%, 9%, and 6% respectively. These results indicate that paeonol has a certain preventive effect on EHP.

[0027] 6. Therapeutic effect of paeonol on EHP: Healthy shrimp were fed with fresh hepatopancreas of EHP-infected shrimp at a feeding amount of 2 g / 10 shrimp and divided into two groups, with 30 shrimp in each group. The culture water of the experimental group contained 10 mg / L of paeonol, and the shrimp were fed three times a day. The culture water containing paeonol was changed every 3 days, denoted as EHP 丹皮酚 . The culture water of the control group contained 0.02% DMSO, denoted as EHP DMSO , and the shrimp were fed three times a day. The culture water containing DMSO was changed every 3 days. At 15, 30, and 45 d, samples were taken to detect the EHP copy number in the shrimp, and the weight of the shrimp in each group was detected at 30 d. The results are as Figure 3 shown.

[0028] As shown in Figure 3 A, after the penaeid shrimps were infected with EHP and treated with paeonol, significant differences in the EHP copy numbers in the penaeid shrimps of the control group and the experimental group were observed on the 15th, 30th, and 45th days after treatment, and the copy number inhibition rates were 52%, 63%, and 84% respectively. As shown in Figure 3 B, in terms of body weight, extremely significant differences were observed after the penaeid shrimps were fed with paeonol for 30 and 45 days, and the weight gain rates were 21% and 24% respectively. These results indicate that paeonol has a good therapeutic effect on EHP.

[0029] 7. Influence of paeonol on the horizontal transmission of EHP: Healthy penaeid shrimps were fed with fresh hepatopancreas of EHP-infected penaeid shrimps at a feeding amount of 2 g / 10 tails. After 7 days, the EHP-infected penaeid shrimps were divided into two groups, with 15 tails in each group. At the same time, 15 healthy penaeid shrimps were placed in each group, and the diseased shrimps and healthy shrimps were separated by a gauze net. The experimental group was cultured with 10 mg / L of paeonol, and the control group was cultured with 0.02 wt% of DMSO in the water body. On the 15th, 30th, and 45th days after commensalism, healthy penaeid shrimps were taken to detect the EHP copy numbers in their bodies.

[0030] The results are as shown in Figure 4 . Paeonol can significantly inhibit the horizontal transmission of EHP. Compared with the control group, when paeonol was present, the inhibition rates of the EHP copy numbers in the healthy shrimps were 57%, 57%, and 64% on the 10th, 20th, and 30th days of commensalism respectively. These results indicate that paeonol has potential applications in preventing the outbreak of EHP.

[0031] Specific Example 2. Preparation of a paeonol preparation for inhibiting Enterocytozoon hepatopenaei.

[0032] The sources of paeonol, Litopenaeus vannamei, and EHP are the same as those in Specific Example 1 above.

[0033] 1. Solvent selection for the paeonol preparation: By observing and comparing the solubility of paeonol in peanut oil, rapeseed oil, ethanol, and acetic acid solvents, it was found that paeonol had no layering or precipitation in peanut oil and ethanol, and the dissolution effect was better. Peanut oil and ethanol were combined in different proportions, and the following 5 groups of experiments were set up to determine the solvent ratio with high solubility and safety.

[0034] Experimental group 1: Weigh 13.0 g of paeonol, 5.0 g of ethanol, and 75.0 g of peanut oil, mix them evenly until the paeonol is completely dissolved, then add 4.0 g of the surfactant Tween-20 and 3.0 g of the transdermal agent water-soluble azone, and mix them evenly to obtain the paeonol preparation.

[0035] Experimental group 2: Weigh 11.0 g of paeonol, 4.0 g of ethanol, 80.0 g of peanut oil, 3.0 g of Tween-20, and 2.0 g of water-soluble azone, and mix them thoroughly to obtain the paeonol preparation. The rest is the same as in experimental group 1.

[0036] Experimental group 3: Weigh 9.0 g of paeonol, 4.0 g of ethanol, 81.0 g of peanut oil, 3.0 g of Tween-20, and 3.0 g of water-soluble azone, and mix them thoroughly to obtain the paeonol preparation. The rest is the same as in experimental group 1.

[0037] Experimental group 4: Weigh 7.0 g of paeonol, 3.0 g of ethanol, 85.0 g of peanut oil, 3.0 g of Tween-20, and 2.0 g of water-soluble azone, and mix them thoroughly to obtain the paeonol preparation. The rest is the same as in experimental group 1.

[0038] Experimental group 5: Weigh 5.0 g of paeonol, 2.0 g of ethanol, 87.0 g of peanut oil, 3.0 g of Tween-20, and 3.0 g of water-soluble azone, and mix them thoroughly to obtain the paeonol preparation. The rest is the same as in experimental group 1.

[0039] Table 1 Results of different solvent ratios of paeonol preparation

[0040] As can be seen from Table 1, the solubility of paeonol in the solvent composed of ethanol and peanut oil (mass ratio range of 1:15 - 1:44) is the highest in experimental group 1, and its solubility exceeds 13 g.

[0041] Anti-EHP activity of paeonol preparation excipients: In this experiment, the effects of other substances (solvent, Tween-20, and water-soluble azone) in the paeonol preparation on the anti-EHP activity of paeonol were determined by comparing the following combinations. Randomly place healthy shrimp in breeding buckets (30 L of breeding water / bucket, 15 shrimp / bucket), add the paeonol preparation of the following experimental groups (total mass of 2 g) to the breeding water and feed the fresh hepatopancreas of EHP-infected shrimp (the EHP concentration in the fresh hepatopancreas of EHP-infected shrimp is 1.6×10 6 copies / mg of tissue, the same in the following experiments), the feeding amount is 2 g / 10 tails, and after incubating for 7 d, detect the EHP copy number in the shrimp.

[0042] Experimental group 1: 13.0 g of paeonol, 5.0 g of ethanol, 75.0 g of peanut oil; Experimental group 2: 13.0 g of paeonol, 5.0 g of ethanol, 75.0 g of peanut oil, 4.0 g of Tween-20 and 3.0 g of water-soluble nitrogen; Experimental group 3: 13.0 g of paeonol, 5.0 g of ethanol, 77.0 g of peanut oil, 3.0 g of Tween-20 and 2.0 g of water-soluble nitrogen; Experimental group 4: 13.0 g of paeonol, 5.0 g of ethanol, 79.0 g of peanut oil, 2.0 g of Tween-20 and 1.0 g of water-soluble nitrogen; Experimental group 5: 5.0 g of ethanol, 95.0 g of peanut oil. The experimental results are shown in Table 2 below. Table 2 Anti-EHP activity results of different formulations of paeonol preparations

[0043] As can be seen from Table 2, Experimental group 5 had no significant anti-EHP activity. Experimental group 2 had the best anti-EHP activity, and Experimental group 4 was the second. The above results indicate that when the total mass ratio of the transdermal agent azone and the surfactant Tween-20 in the paeonol preparation is less than or equal to 7%, it can improve its anti-EHP activity.

[0044] 3. Anti-EHP activity of different concentrations of paeonol preparations: Randomly place healthy shrimp in breeding barrels (30 L of breeding water / barrel, 15 shrimp / barrel). The experiment was set up with four groups of breeding water with concentrations of 0 mg / L, 10 mg / L, 20 mg / L, and 30 mg / L of paeonol preparations (13.0 g of paeonol, 5.0 g of ethanol, 75.0 g of peanut oil, 4.0 g of Tween-20, and 3.0 g of water-soluble nitrogen). Each group was fed the fresh hepatopancreas of EHP-infected shrimp, and the water containing the same concentration of paeonol preparation was changed every 3 days. At the 45th day, the hepatopancreas of the shrimp in each group was collected to detect the EHP copy number; at the same time, 10 shrimp were taken from each group and weighed to compare the differences. The results are shown in Table 3 below. Table 3 Anti-EHP activity of paeonol preparations

[0045] Note: a-b in the table: Different letters indicate significant differences between different treatment groups ( p <0.05).

[0046] As can be seen from Table 3, adding different doses of paeonol preparations to the breeding water can inhibit the replication of EHP in shrimp and promote their weight gain. When the addition amount of the paeonol preparation is 30 mg / L, the inhibition rate of EHP replication in shrimp is 82%, and the weight of the shrimp is significantly increased compared with the control group. In summary, the paeonol preparation can effectively prevent and control EHP infection.

[0047] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions or substitutions made by those of ordinary skill in the art within the scope of the essence of the present invention shall also fall within the protection scope of the present invention.

Claims

1. Application of paeonol in preparing an inhibitor of Enterocytozoon hepatopenaei.

2. Application of paeonol in preparing a drug for preventing and / or treating Enterocytozoon hepatopenaei disease.

3. A paeonol preparation for inhibiting Enterocytozoon hepatopenaei, characterized in that It consists of the following raw materials and their mass percentages: paeonol 5 - 13%, ethanol 3 - 5%, Tween-20 2 - 4%, water-soluble azone 1 - 3%, and the remaining part is peanut oil.

4. The paeonol preparation for inhibiting Enterocytozoon hepatopenaei according to claim 3, characterized in that The paeonol preparation consists of the following raw materials and their mass percentages: paeonol 13.0%, ethanol 5.0%, peanut oil 75.0%, Tween-20 4.0%, and water-soluble azone 3.0%.

5. The paeonol preparation for inhibiting Enterocytozoon hepatopenaei according to claim 3, characterized in that The paeonol preparation consists of the following raw materials and their mass percentages: paeonol 13.0%, ethanol 5.0%, peanut oil 77.0%, Tween-20 3.0%, and water-soluble azone 2.0%.

6. The paeonol preparation for inhibiting Enterocytozoon hepatopenaei according to claim 3, wherein The paeonol preparation consists of the following raw materials and their mass percentages: paeonol 13.0%, ethanol 5.0%, peanut oil 79.0%, Tween-20 2.0%, and water-soluble azone 1.0%.

7. A method for preparing the paeonol preparation for inhibiting Enterocytozoon hepatopenaei according to any one of claims 3-6, characterized in that It includes the following steps: weigh paeonol, Tween-20, water-soluble azone, ethanol, and peanut oil for standby; mix paeonol, ethanol, and peanut oil until paeonol is completely dissolved, then add Tween-20 and water-soluble azone, and mix evenly to obtain the paeonol preparation.

8. Use of the paeonol preparation for inhibiting Enterocytozoon hepatopenaei according to any one of claims 3-6 in the culture of penaeid shrimps, characterized in that, The addition concentration of the paeonol preparation in the shrimp culture water is 30 mg / L.

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