Anti-enterocytozoon hepatopenaei traditional Chinese medicine compound as well as preparation method and application thereof

Abstract

The invention discloses an anti-enterocytozoon hepatopenaei traditional Chinese medicine compound and a preparation method and application thereof.The anti-enterocytozoon hepatopenaei traditional Chinese medicine compound is characterized by being prepared from, by weight, 10-20 parts of sophora alopecuroide alcohol extract, 50-75 parts of cortex fraxini alcohol extract and 10-30 parts of fructus evodiae alcohol extract.The preparation method comprises the following steps that dried and smashed sophora alopecuroide, cortex fraxini and fructus evodiae are weighed; respectively dissolving and extracting by using absolute ethyl alcohol to obtain alcohol extracts; the preparation method comprises the following steps: weighing the traditional Chinese medicine alcohol extract in parts by weight, uniformly mixing to obtain the anti-enterocytozoon hepatopenaei traditional Chinese medicine compound, and further provides application of the anti-enterocytozoon hepatopenaei traditional Chinese medicine compound, the sophora alopecuroide alcohol extract, the ash bark alcohol extract and the fructus evodiae alcohol extract in preparation of an enterocytozoon hepatopenaei inhibitor. The weight gain rate of prawns is increased, economic losses caused by outbreak of enterocytozoon hepatopenaei to prawn culture can be effectively reduced, and the compound is low in cost and simple in use method.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and in particular to a traditional Chinese medicine compound against Enterocytozoon hepatopenaei, its preparation method and application. Background Art

[0002] Enterocytozoon hepatopenaei ( Enterocytozoon hepatopenaei , abbreviated as EHP) is an obligate intracellular parasite, and the Enterocytozoon hepatopenaei disease it causes is listed as a class II animal disease by the Ministry of Agriculture of China. EHP causes a decline in the feeding ability of shrimp, slow or even stagnant growth, leading to empty consumption of 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 the sustainable development of aquaculture. The main prevention and control strategy for aquatic parasitic diseases is to use drugs. However, existing anti-parasitic fishery drugs such as pyrethroids, trichlorfon and mebendazole not only have limited efficacy, but also problems such as food and environmental safety limit their application in aquaculture. Traditional Chinese medicine has the advantages of natural safety, small toxic and side effects, low residue, and not easy to produce drug resistance, and has attracted much attention in the prevention and control of aquatic animal diseases. Research shows that traditional Chinese medicines such as Sophora alopecuroides, Cortex Fraxini and Evodia rutaecarpa have significant effects in aspects such as antibacterial, insecticidal, antioxidant and immune regulation. At present, there is no research on the anti-EHP effect of the above-mentioned traditional Chinese medicine ethanol extracts and their compounds. Exploring the anti-EHP activity of the above-mentioned traditional Chinese medicine ethanol extracts and their compounds has important practical significance for controlling the outbreak of Enterocytozoon hepatopenaei disease. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a traditional Chinese medicine compound against Enterocytozoon hepatopenaei with improved anti-Enterocytozoon hepatopenaei activity in penaeid shrimp, its preparation method and application.

[0004] The technical solution adopted by the present invention to solve the above technical problem is: a traditional Chinese medicine compound against Enterocytozoon hepatopenaei, which is composed of the following raw materials and their weight parts: 10-20 parts of Sophora alopecuroides ethanol extract, 55-75 parts of Cortex Fraxini ethanol extract, and 10-30 parts of Evodia rutaecarpa ethanol extract.

[0005] Preferably, it is composed of the following raw materials and their weight parts: 10 parts of Sophora alopecuroides extract, 60 parts of Cortex Fraxini ethanol extract, and 30 parts of Evodia rutaecarpa ethanol extract.

[0006] Preferably, it is composed of the following raw materials and their weight parts: 20 parts of Sophora alopecuroides extract, 55 parts of Cortex Fraxini ethanol extract, and 25 parts of Evodia rutaecarpa ethanol extract.

[0007] Preferably, it is composed of the following raw materials and their weight parts: 15 parts of Sophora alopecuroides extract, 75 parts of Cortex Fraxini ethanol extract, and 10 parts of Evodia rutaecarpa ethanol extract.

[0008] Further, the preparation methods of the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa are as follows: Take Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa, dry and crush them, sieve them through a 100-mesh sieve, weigh 50 g each and add 300 mL of absolute ethanol, place them in an ultrasonic cleaner for ultrasonic dissolution for 10 min, then place them in a water bath at 60 °C and heat for 30 min, repeat the above steps 3 times, add 150 mL of absolute ethanol each time, concentrate the absolute ethanol leaching solution by rotary evaporation, and finally dry it in an oven to obtain the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa respectively.

[0009] The present invention also provides a preparation method of the above-mentioned traditional Chinese medicine compound against Enterocytozoon hepatopenaei, including the following steps: (1) Weigh the dried and crushed Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa, and dissolve and extract them respectively with absolute ethanol to obtain ethanol extracts; (2) Weigh 10 - 20 parts by weight of the ethanol extract of Sophora alopecuroides, 50 - 75 parts by weight of the ethanol extract of Cortex Fraxini, and 10 - 30 parts by weight of the ethanol extract of Evodia rutaecarpa, mix them evenly to obtain the traditional Chinese medicine compound against EHP.

[0010] The present invention also provides the application of the above-mentioned traditional Chinese medicine compound against Enterocytozoon hepatopenaei in the preparation of an Enterocytozoon hepatopenaei inhibitor.

[0011] The present invention also provides the application of the above-mentioned ethanol extract of Sophora alopecuroides in the preparation of an Enterocytozoon hepatopenaei inhibitor, and the addition amount of the ethanol extract of Sophora alopecuroides in the penaeid shrimp culture water is 10 - 40 mg / L.

[0012] The present invention also provides the application of the above-mentioned ethanol extract of Cortex Fraxini in the preparation of an Enterocytozoon hepatopenaei inhibitor, and the addition amount of the ethanol extract of Cortex Fraxini in the penaeid shrimp culture water is 10 - 80 mg / L.

[0013] The present invention also provides the application of the above-mentioned ethanol extract of Evodia rutaecarpa in the preparation of an Enterocytozoon hepatopenaei inhibitor, and the addition amount of the ethanol extract of Evodia rutaecarpa in the penaeid shrimp culture water is 10 - 80 mg / L.

[0014] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a traditional Chinese medicine compound against Enterocytozoon hepatopenaei, its preparation method and application. After drying and crushing Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa, the effective components are extracted with absolute ethanol, and the ethanol extracts are weighed according to the ratio and mixed evenly to obtain the traditional Chinese medicine compound against EHP, which can effectively block the outbreak of EHP. The anti-EHP effect of the traditional Chinese medicine compound adopted is more significant than that of using the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa alone, and its preparation and use methods are simple, natural and pollution-free, and can effectively reduce the economic losses caused by the outbreak of EHP to penaeid shrimp culture. The compound has a low cost and a simple use method, which is conducive to popularization and application. Description of the Drawings

[0015] Figure 1Effect of ethanol extracts of traditional Chinese medicines on the survival rate of Litopenaeus vannamei, where A is the ethanol extract of Sophora alopecuroides, B is the ethanol extract of Cortex Fraxini, and C is the ethanol extract of Evodia rutaecarpa; Figure 2 Analysis of the anti-EHP activity of ethanol extracts of traditional Chinese medicines; Figure 3 Treatment of Litopenaeus vannamei infected with EHP with the ethanol extract of Sophora alopecuroides, where A is the effect of the ethanol extract of Sophora alopecuroides on the EHP copy number, and B is the effect of the ethanol extract of Sophora alopecuroides on the body weight of Litopenaeus vannamei infected with EHP; Figure 4 Treatment of Litopenaeus vannamei infected with EHP with the ethanol extract of Cortex Fraxini, where A is the effect of the ethanol extract of Cortex Fraxini on the EHP copy number, and B is the effect of the ethanol extract of Cortex Fraxini on the body weight of Litopenaeus vannamei infected with EHP; Figure 5 Treatment of Litopenaeus vannamei infected with EHP with the ethanol extract of Evodia rutaecarpa, where A is the effect of the ethanol extract of Evodia rutaecarpa on the EHP copy number, and B is the effect of the ethanol extract of Evodia rutaecarpa on the body weight of Litopenaeus vannamei infected with EHP; Figure 6 Treatment of Litopenaeus vannamei infected with EHP with Compound A, where A is the effect of Compound A on the EHP copy number, and B is the effect of Compound A on the body weight of Litopenaeus vannamei infected with EHP; Figure 7 Treatment of Litopenaeus vannamei infected with EHP with Compound B, where A is the effect of Compound B on the EHP copy number, and B is the effect of Compound B on the body weight of Litopenaeus vannamei infected with EHP; Figure 8 Treatment of Litopenaeus vannamei infected with EHP with Compound C, where A is the effect of Compound C on the EHP copy number, and B is the effect of Compound C on the body weight of Litopenaeus vannamei infected with EHP. Detailed implementation mode

[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, the anti-EHP activities of the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, Evodia rutaecarpa and their compounds were analyzed using Litopenaeus vannamei as the object. Among them, Sophora alopecuroides, Cortex Fraxini, Evodia rutaecarpa, dimethyl sulfoxide (DMSO), and absolute ethanol were all provided by Shanghai Aladdin Biochemical Technology Co., Ltd., and EHP and Litopenaeus vannamei were provided by the Zhejiang Institute of Marine Fisheries.

[0018] Preparation of the mother liquor of the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa: Take Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa, dry and crush them. After sieving through a 100-mesh sieve, weigh 50 g each and add 300 mL of absolute ethanol. Place them in an ultrasonic cleaner for ultrasonic dissolution for 10 min, then place them in a water bath at 60 °C and heat for 30 min. Repeat the above steps 3 times, adding 150 mL of absolute ethanol each time. Take the above absolute ethanol extracts and perform rotary evaporation and concentration respectively. Finally, place them in an oven to dry to obtain the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa. Dissolve the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa separately with DMSO to prepare a mother liquor with a final concentration of 50000 mg / L and store it at 4 °C for later use.

[0019] Detection of EHP copy number: Extract shrimp DNA using a rapid extraction kit for genomic DNA from marine animal tissues (Tiangen). After detecting the DNA concentration (adjusting 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 in SEQ ID NO.1: 5'-GTAGCGGAACGGATAGGG-3', ENF 185 The nucleotide sequence of the reverse amplification primer is shown in SEQ ID NO.2: 5'-CCAGCATTGTCGGCATAG-3'. The qPCR amplification reaction system is as follows: 2 × Es Taq MasterMix 7.5 μL, 10 μM forward amplification primer 0.3 μL, 10 μM reverse amplification primer 0.3 μL, template DNA 1 μL, ddH2O 5.9 μL. 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, 35 cycles; 72 °C for 10 min. Calculate the EHP copy number from the qPCR results according to the standard curve made with the pMD19T-ENF 185 standard product.

[0020] Specific Example 1. Toxicity experiments of the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa.

[0021] Take healthy shrimp and divide them into 5 groups, with 15 shrimp in each group. By the immersion method, the experiment is set such that the concentrations of the ethanol extract of Sophora alopecuroides in the culture water are 0, 10, 20, 40, 80, and 160 mg / L respectively, and the control group is that the mass concentration of DMSO in the culture water is 0.02%. After 72 h, count the survival rate of the shrimp, and determine the safe concentration of the ethanol extract of Sophora alopecuroides according to the survival rate of the shrimp. The results are as Figure 1As shown in Figure A, when the addition concentration of the ethanol extract of Sophora alopecuroides in the shrimp culture water is 40 mg / L, it has no significant effect on the survival of shrimp and can be used for subsequent experiments.

[0022] Similarly, the concentrations of the ethanol extract of Cortex Fraxini in the culture water were set to 0, 10, 20, 40, 80, and 160 mg / L respectively in the experiment, and the control group was the culture water with a DMSO mass concentration of 0.02%. The survival rate of shrimp was counted after 72 h, and the results are as Figure 1 shown in Figure B. When the addition concentration of the ethanol extract of Cortex Fraxini in the shrimp culture water is 80 mg / L, it has no significant effect on the survival of shrimp and can be used for subsequent experiments.

[0023] Similarly, the concentrations of the ethanol extract of Evodia rutaecarpa in the culture water were set to 0, 10, 20, 40, 80, and 160 mg / L respectively in the experiment, and the control group was the culture water with a DMSO mass concentration of 0.02%. The survival rate of shrimp was counted after 72 h, and the results are as Figure 1 shown in Figure C. When the addition concentration of the ethanol extract of Evodia rutaecarpa in the shrimp culture water is 80 mg / L, it has no significant effect on the survival of shrimp and can be used for subsequent experiments.

[0024] Specific Example 2: Analysis of the anti-EHP activities of the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa.

[0025] 1. Preliminary analysis of the anti-EHP activities of the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa: In the experiment, three groups of culture water were set with the addition concentration of the ethanol extract of Sophora alopecuroides being 40 mg / L, the ethanol extract of Cortex Fraxini being 80 mg / L, and the ethanol extract of Evodia rutaecarpa being 80 mg / L. The control group was the culture water containing 0.02 wt% DMSO. Each group was fed with the fresh hepatopancreas of shrimp infected with EHP (the EHP concentration in the fresh hepatopancreas of shrimp infected with EHP was 1.6×10 6 copies / mg tissue, the same in the following experiments), the feeding amount was 2 g / 10 tails, and each group had 10 shrimp. On the 7th day after infection, samples were taken and the EHP copy number was detected by qPCR.

[0026] The results are as Figure 2 shown. Compared with the control group, the ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa can all significantly reduce the EHP copy number in shrimp, and the inhibition rates of the EHP copy number are 50%, 47%, and 58% respectively, indicating that the above-mentioned ethanol extracts all have a certain effect on inhibiting the proliferation of EHP.

[0027] 2. Anti-EHP activity analysis of ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa: Healthy shrimp were fed with fresh hepatopancreas of EHP-infected shrimp at a feeding rate of 2 g / 10 tails and divided into two groups, with 30 shrimp in each group. After 7 days, they were fed commercial feeds containing 0.5 wt% ethanol extract of Sophora alopecuroides, 0.5 wt% ethanol extract of Cortex Fraxini, and 0.5 wt% ethanol extract of Evodia rutaecarpa, respectively. The control group was fed commercial feed containing 0.5 wt% DMSO, and the feed was given three times a day, 1 g each time. On the 10th, 20th, and 30th days after feeding, the hepatopancreas of the shrimp was taken to detect the EHP copy number, and the weight of the shrimp in each group was also weighed.

[0028] The results are as Figure 3 shown in A below. Compared with the control group, the EHP copy number in the shrimp decreased significantly after 20 and 30 days of feeding the feed containing the ethanol extract of Sophora alopecuroides; as Figure 3 shown in B below, in terms of the weight index, compared with the control group, the weight of the EHP-infected shrimp increased by 11% after 30 days of feeding, but there was no significant difference.

[0029] The results are as Figure 4 shown in A below. Compared with the control group, the EHP copy number in the shrimp decreased significantly after 10, 20, and 30 days of feeding the feed containing the ethanol extract of Cortex Fraxini. As Figure 4 shown in B below, in terms of the weight index, compared with the control group, the weight of the EHP-infected shrimp increased by 14% after 30 days of feeding the feed containing the ethanol extract of Cortex Fraxini.

[0030] The results are as Figure 5 shown in A below. Compared with the control group, the EHP copy number in the shrimp decreased significantly after 20 and 30 days of feeding the feed containing the ethanol extract of Evodia rutaecarpa. As Figure 5 shown in B below, in terms of the weight index, compared with the control group, the weight of the EHP-infected shrimp increased by 12% after 30 days of feeding the feed containing the ethanol extract of Evodia rutaecarpa. The above results indicate that these ethanol extracts of traditional Chinese medicines have the potential to be applied in the treatment of EHP-infected shrimp.

[0031] Specific Example 3. Anti-EHP activity analysis of the compound of ethanol extracts of Sophora alopecuroides, Cortex Fraxini, and Evodia rutaecarpa.

[0032] First, prepare the compound formulations according to the weight ratio: Compound A (extract of Sophora alopecuroides L. by ethanol extraction: extract of Cortex Fraxini by ethanol extraction: extract of Evodia rutaecarpa (Juss.) Benth. by ethanol extraction = 10:60:30), Compound B (extract of Sophora alopecuroides L. by ethanol extraction: extract of Cortex Fraxini by ethanol extraction: extract of Evodia rutaecarpa (Juss.) Benth. by ethanol extraction = 20:55:25), and Compound C (extract of Sophora alopecuroides L. by ethanol extraction: extract of Cortex Fraxini by ethanol extraction: extract of Evodia rutaecarpa (Juss.) Benth. by ethanol extraction = 15:75:10). Feed the fresh hepatopancreas of EHP-infected healthy shrimp to healthy shrimp at a feeding rate of 2 g / 10 tails, and divide them into two groups with 30 shrimp in each group. After 7 days, feed commercial feeds containing 0.5 wt% Compound A, 0.5 wt% Compound B, and 0.5 wt% Compound C respectively, and feed the control group with commercial feed containing 0.5 wt% DMSO, three times a day, 1 g each time. On the 10th, 20th, and 30th days after feeding, take the hepatopancreas of the shrimp to detect the EHP copy number, and at the same time weigh the shrimp in each group.

[0033] The results are as Figure 6 shown in Figure 7 Figure Figure 8 A. After feeding the feeds containing Compound A, Compound B, and Compound C for 10, 20, and 30 days, the EHP copy number in the shrimp decreased significantly. As Figure 6 shown in Figure 7 Figure Figure 8 B. In terms of the body weight index, after feeding the feeds containing Compound A, Compound B, and Compound C for 30 days respectively, the EHP-infected shrimp increased their weights by 22%, 18%, and 16% respectively, and there was a significant difference between the group fed with the feed containing Compound A and the control group. The above results indicate that Compound A has the best therapeutic effect on EHP-infected shrimp.

[0034] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Any 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. A traditional Chinese medicine compound against Enterocytozoon hepatopenaei, characterized in that It consists of the following raw materials and their parts by weight: 10-20 parts of sophora alopecuroides ethanol extract, 50-75 parts of fraxinus rhynchophylla ethanol extract, and 10-30 parts of evodia rutaecarpa ethanol extract.

2. The traditional Chinese medicine compound against Enterocytozoon hepatopenaei according to claim 1, characterized in that It consists of the following raw materials and their parts by weight: 10 parts of sophora alopecuroides ethanol extract, 60 parts of fraxinus rhynchophylla ethanol extract, and 30 parts of evodia rutaecarpa ethanol extract.

3. The traditional Chinese medicine compound against Enterocytozoon hepatopenaei according to claim 1, characterized in that It consists of the following raw materials and their parts by weight: 20 parts of sophora alopecuroides extract, 55 parts of fraxinus rhynchophylla ethanol extract, and 25 parts of evodia rutaecarpa ethanol extract.

4. The traditional Chinese medicine compound against Enterocytozoon hepatopenaei according to claim 1, wherein It consists of the following raw materials and their parts by weight: 15 parts of sophora alopecuroides extract, 75 parts of fraxinus rhynchophylla ethanol extract, and 10 parts of evodia rutaecarpa ethanol extract.

5. A traditional Chinese medicine compound against Enterocytozoon hepatopenaei according to any one of claims 1-4, characterized in that The preparation methods of the sophora alopecuroides ethanol extract, fraxinus rhynchophylla ethanol extract, and evodia rutaecarpa ethanol extract are as follows: Take sophora alopecuroides, fraxinus rhynchophylla, and evodia rutaecarpa, dry and crush them, sieve them through a 100-mesh sieve, then weigh 50 g each and add 300 mL of absolute ethanol, place them in an ultrasonic cleaner for ultrasonic dissolution for 10 min, then place them in a water bath at 60 °C and heat for 30 min. Repeat the above steps 3 times, adding 150 mL of absolute ethanol each time. Rotate and evaporate and concentrate the absolute ethanol leaching solution, and finally dry it in an oven to obtain the sophora alopecuroides ethanol extract, fraxinus rhynchophylla ethanol extract, and evodia rutaecarpa ethanol extract respectively.

6. A preparation method of the traditional Chinese medicine compound against Enterocytozoon hepatopenaei according to claim 5, characterized in that It includes the following steps: (1) Weigh the dried and crushed sophora alopecuroides, fraxinus rhynchophylla, and evodia rutaecarpa, and dissolve and extract them respectively with absolute ethanol to obtain ethanol extracts; (2) Weigh 10-20 parts of sophora alopecuroides ethanol extract, 50-75 parts of fraxinus rhynchophylla ethanol extract, and 10-30 parts of evodia rutaecarpa ethanol extract by weight, mix them evenly to obtain the anti-EHP traditional Chinese medicine compound.

7. Use of the anti-Enterocytozoon hepatopenaei traditional Chinese medicine compound according to claim 1 in the preparation of an Enterocytozoon hepatopenaei inhibitor.

8. Use of the ethanol extract of Sophora alopecuroides described in claim 1 in the preparation of an Enterocytozoon hepatopenaei inhibitor, characterized in that: The addition amount of the sophora alopecuroides ethanol extract in the shrimp culture water is 10-40 mg / L.

9. Use of the ethanol extract of Cortex Fraxini described in claim 1 in the preparation of an inhibitor of Enterocytozoon hepatopenaei, characterized in that: The addition amount of the fraxinus rhynchophylla ethanol extract in the shrimp culture water is 10-80 mg / L.

10. Use of the ethanol extract of Evodia rutaecarpa described in claim 1 in the preparation of an inhibitor of Enterocytozoon hepatopenaei, characterized in that: The addition amount of the evodia rutaecarpa ethanol extract in the shrimp culture water is 10-80 mg / L.

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