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 invention relates to the technical field of biomedicine, and in particular to a traditional Chinese medicine compound for resisting shrimp hepatoenteric cysts, and a preparation method and application thereof. Background Art

[0002] Shrimp Hepatocellular Carcinoma Enterocytozoon hepatopenaei EHP (EHP for short) is an obligate intracellular parasite. The shrimp hepatoenterozoan disease it causes is classified as a Class II animal disease. EHP causes shrimps to have a reduced feeding capacity, slow growth or even stagnation, leading to feed consumption and easily causing secondary bacterial diseases. It has the characteristics of multiple infection vectors, wide distribution and strong transmission ability, which seriously threatens the sustainable development of aquaculture. The main prevention and control strategy for aquatic parasitic diseases is to use drugs. However, existing anti-insect fishery drugs such as pyrethroids, trichlorfon and mebendazole not only have limited efficacy, but also have food and environmental safety issues that further restrict their application in aquaculture. Traditional Chinese medicine has the advantages of natural safety, low toxicity and side effects, low residue, and low resistance to drug resistance, and has attracted much attention in the prevention and control of aquatic animal diseases. Studies have shown that the traditional Chinese medicines bitter beans, Qinpi and Evodia rutaecarpa have significant effects in antibacterial, insecticidal, antioxidant and immunomodulatory aspects. At present, there is no research on the anti-EHP activity of the above-mentioned Chinese herbal alcohol extracts and their compounds. Exploring the anti-EHP activity of the above-mentioned Chinese herbal alcohol extracts and their compounds has important practical significance for controlling the outbreak of shrimp hepatoenterocytosis. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a traditional Chinese medicine compound capable of improving the anti-shrimp hepatoenterocyte activity and anti-shrimp hepatoenterocyte parasite activity of shrimp, and a preparation method and application thereof.

[0004] The technical solution adopted by the present invention to solve the above technical problems is: a traditional Chinese medicine compound for resisting shrimp hepatoenteric cytozoon, which is composed of the following raw materials and their weight proportions: 10-20 parts of sophora alopecuroides ethanol extract, 55-75 parts of frassilis cortex ethanol extract and 10-30 parts of evodia rutaecarpa ethanol extract.

[0005] Preferably, it is composed of the following raw materials and their weight proportions: 10 parts of Sophora alopecuroides extract, 60 parts of Fraxinus fraseri alcohol extract and 30 parts of Evodia rutaecarpa alcohol extract.

[0006] Preferably, it is composed of the following raw materials and their weight proportions: 20 parts of Sophora alopecuroides extract, 55 parts of Fraxinus fraseri alcohol extract and 25 parts of Evodia rutaecarpa alcohol extract.

[0007] Preferably, it is composed of the following raw materials and their weight proportions: 15 parts of Sophora alopecuroides extract, 75 parts of Fraxinus fraseri alcohol extract and 10 parts of Evodia rutaecarpa alcohol extract.

[0008] Furthermore, the preparation method of the alcohol extract of Sophora alopecuroides, the alcohol extract of Fraxinus frasii and the alcohol extract of Evodia rutaecarpa is as follows: Sophora alopecuroides, Fraxinus frasii and Evodia rutaecarpa are dried and crushed, 50 g of each are weighed and added with 300 mL of anhydrous ethanol after sieving through a 100-mesh sieve, the extracts are placed in an ultrasonic cleaner for ultrasonic dissolution for 10 min, and then heated in a 60°C water bath for 30 min, the above steps are repeated 3 times, 150 mL of anhydrous ethanol is added each time, the anhydrous ethanol extract is concentrated by rotary evaporation, and finally dried in an oven to obtain the alcohol extract of Sophora alopecuroides, the alcohol extract of Fraxinus frasii and the alcohol extract of Evodia rutaecarpa, respectively.

[0009] The present invention also provides a method for preparing the above-mentioned anti-shrimp hepato-enteric cytopenia Chinese medicine compound, comprising the following steps: (1) Weigh the dried and crushed bitter bean, qinpi and rutaecarpa, and use anhydrous ethanol to dissolve and extract to obtain ethanol extracts; (2) Weigh 10-20 parts of the alcohol extract of Sophora alopecuroides, 50-75 parts of the alcohol extract of Fragrant Cortex and 10-30 parts of the alcohol extract of Evodia rutaecarpa by weight, and mix them evenly to obtain the anti-EHP Chinese medicine compound.

[0010] The present invention also provides application of the above-mentioned traditional Chinese medicine compound for resisting shrimp hepatoenteric cytozoon in preparing shrimp hepatoenteric cytozoon inhibitor.

[0011] The present invention also provides the use of the alcohol extract of Sophora alopecuroides in the preparation of shrimp hepato-enteric cytozoon inhibitors, wherein the alcohol extract of Sophora alopecuroides is added in an amount of 10-40 mg / L in shrimp culture water.

[0012] The present invention also provides the use of the alcohol extract of Fraxinus fraxinus in the preparation of shrimp hepatoenteric cytozoon inhibitors, wherein the alcohol extract of Fraxinus fraxinus is added in an amount of 10-80 mg / L in shrimp breeding water.

[0013] The present invention also provides the use of the above-mentioned Evodia rutaecarpa alcohol extract in the preparation of shrimp hepato-enteric cytozoon inhibitors, wherein the addition amount of the above-mentioned Evodia rutaecarpa alcohol extract in shrimp breeding water is 10-80 mg / L.

[0014] Compared with the prior art, the invention has the advantages that: the invention discloses a Chinese medicine compound for resisting hepatoenteric cytozoon of shrimp, a preparation method thereof and an application thereof, wherein the effective components are extracted by anhydrous ethanol after drying and crushing bitter bean, qinpi and rutaecarpa, and the alcohol extract is weighed in proportion and mixed to obtain the Chinese medicine compound for resisting EHP, which can effectively block the outbreak of EHP, and the anti-EHP effect of the Chinese medicine compound is more significant than that of the alcohol extract of bitter bean, qinpi and rutaecarpa used alone, and the preparation and use methods thereof are simple, natural and pollution-free, and can effectively reduce the economic losses caused by the outbreak of EHP to shrimp farming. The compound has low cost, simple use method and is conducive to popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The effect of Chinese medicine alcohol extracts on the survival rate of shrimp, where A is alcohol extract of Sophora alopecuroides, B is alcohol extract of Fraxinus fraseri, and C is alcohol extract of Evodia rutaecarpa; Figure 2 To analyze the anti-EHP activity of Chinese herbal medicine alcohol extracts; Figure 3 The treatment of EHP-infected shrimps with alcohol extract of Sophora alopecuroides, A is the effect of alcohol extract of Sophora alopecuroides on the number of EHP copies, B is the effect of alcohol extract of Sophora alopecuroides on the body weight of EHP-infected shrimps; Figure 4 The treatment of EHP-infected shrimps with the alcohol extract of Fraxinus fraxinus, where A is the effect of the alcohol extract of Fraxinus fraxinus on the number of EHP copies, and B is the effect of the alcohol extract of Fraxinus fraxinus on the body weight of EHP-infected shrimps; Figure 5 The treatment of Evodia rutaecarpa ethanol extract on shrimp infected with EHP, where A is the effect of Evodia rutaecarpa ethanol extract on the EHP copy number, and B is the effect of Evodia rutaecarpa ethanol extract on the body weight of shrimp infected with EHP; Figure 6 is the treatment of compound A on shrimp infected with EHP, where A is the effect of compound A on the number of EHP copies, and B is the effect of compound A on the body weight of shrimp infected with EHP; Figure 7 is the treatment of compound B on shrimp infected with EHP, where A is the effect of compound B on the number of EHP copies, and B is the effect of compound B on the body weight of shrimp infected with EHP; Figure 8 The treatment of compound C on shrimp infected with EHP, where A is the effect of compound C on the copy number of EHP, and B is the effect of compound C on the body weight of shrimp infected with EHP. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the accompanying drawings.

[0017] To illustrate the essence of the present invention, the anti-EHP activity of alcohol extracts of Sophora alopecuroides, Cortex Fraxini and Evodia rutaecarpa and their compound were analyzed with Litopenaeus vannamei as the object. Sophora alopecuroides, Cortex Fraxini, Evodia rutaecarpa, dimethyl sulfoxide (DMSO) and anhydrous ethanol were all provided by Shanghai Aladdin Biochemical Technology Co., Ltd., and EHP and Litopenaeus vannamei were provided by Zhejiang Marine Aquaculture Research Institute.

[0018] Preparation of mother liquor of alcohol extract of bitter bean, qinpi and rutaecarpa: dry and crush bitter bean, qinpi and rutaecarpa, sieve through 100 mesh sieve, weigh 50 g respectively, add 300 mL of anhydrous ethanol, place in ultrasonic cleaning instrument for ultrasonic dissolution for 10 min, then place in 60℃ water bath for 30 min, repeat the above steps 3 times, add 150 mL of anhydrous ethanol each time. Take the above anhydrous ethanol extracts respectively, carry out rotary evaporation and concentration, and finally place in oven to dry to obtain alcohol extracts of bitter bean, qinpi and rutaecarpa, and fully dissolve the alcohol extracts of bitter bean, qinpi and rutaecarpa in DMSO, prepare the mother liquor with a final concentration of 50000 mg / L, and store at 4℃ for use.

[0019] EHP copy number detection: Shrimp DNA was extracted using a marine animal tissue genomic DNA rapid extraction kit (Tian Gen), and the DNA concentration (adjusted to a final concentration of 100 ng / μL) and purity were detected by ultra-micro spectrophotometer before qPCR amplification experiments. qPCR amplification primers included 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', and 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, ddH 2 O 5.9 μL. The qPCR amplification reaction program is as follows: 95℃ for 5 min; 95℃ for 30 s, 55.5℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 10 min. 185 The standard curve made from the standard product was used to convert the qPCR results to obtain the EHP copy number.

[0020] Specific Example 1: Toxicity experiment of ethanol extracts of Sophora alopecuroides, Fraxinus fraseri and Evodia rutaecarpa.

[0021] Healthy shrimps were divided into 5 groups, 15 in each group. The experiment was conducted by immersion. The concentrations of alcohol extract of Sophora alopecuroides in the aquaculture water were 0, 10, 20, 40, 80 and 160 mg / L, respectively. The DMSO concentration in the aquaculture water of the control group was 0.02%. The survival rate of shrimps was calculated after 72 hours, and the safe concentration of alcohol extract of Sophora alopecuroides was determined based on the survival rate of shrimps. The results are shown in Figure 1As shown in Figure A, when the concentration of the alcohol extract of Sophora alopecuroides added to the shrimp culture water was 40 mg / L, it had no significant effect on the survival of the shrimp and could be used for subsequent experiments.

[0022] Similarly, the concentrations of the alcohol extract of Fraxinus chinensis in the aquaculture water were set at 0, 10, 20, 40, 80 and 160 mg / L respectively, and the DMSO mass concentration in the aquaculture water of the control group was 0.02%. The survival rate of the shrimp was calculated at 72 h. The results are as follows Figure 1 As shown in B, when the concentration of the alcohol extract of Fraxinus chinensis added to the shrimp culture water was 80 mg / L, it had no significant effect on the survival of the shrimp and could be used for subsequent experiments.

[0023] In the same experiment, the concentrations of the alcohol extract of Evodia rutaecarpa in the aquaculture water were 0, 10, 20, 40, 80 and 160 mg / L respectively, and the DMSO mass concentration in the aquaculture water of the control group was 0.02%. The survival rate of the shrimp was calculated after 72 hours. The results are as follows Figure 1 As shown in C, when the concentration of Evodia rutaecarpa ethanol extract added to shrimp culture water was 80 mg / L, it had no significant effect on shrimp survival and could be used for subsequent experiments.

[0024] Specific Example 2: Analysis of the anti-EHP activity of the ethanol extracts of Sophora alopecuroides, Fraxinus frasii and Evodia rutaecarpa.

[0025] 1. Preliminary analysis of the anti-EHP activity of alcohol extracts of Sophora alopecuroides, Fraxinus fraxinus and Evodia rutaecarpa: The experiment was set up with three groups of aquaculture water added with 40 mg / L alcohol extract of Sophora alopecuroides, 80 mg / L alcohol extract of Fraxinus fraxinus and 80 mg / L alcohol extract of Evodia rutaecarpa. The control group was aquaculture water containing 0.02wt% DMSO. Each group was fed with fresh hepatopancreas of EHP-infected shrimp (the EHP concentration in the fresh hepatopancreas of EHP-infected shrimp was 1.6×10 6 The feed amount was 2 g / 10 shrimps, and each group had 10 shrimps. On the 7th day of infection, samples were taken and the EHP copy number was detected by qPCR.

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

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

[0028] The results are as follows Figure 3 As shown in Figure A, compared with the control group, the EHP copy number in shrimp was significantly reduced after feeding the diet containing alcohol extract of Sophora alopecuroides for 20 and 30 days; Figure 3 As shown in B, in terms of body weight, the EHP-infected shrimp gained 11% after 30 days of feeding compared with the control group, but there was no significant difference.

[0029] The results are as follows Figure 4 As shown in Figure A, compared with the control group, the EHP copy number in shrimp was significantly reduced after feeding the diet containing the alcohol extract of Fraxinus fraxini for 10, 20 and 30 days. Figure 4 As shown in B, in terms of body weight, the EHP-infected shrimp gained 14% after being fed with a diet containing alcohol extract of Fraxinus fraxini for 30 days compared with the control group.

[0030] The results are as follows Figure 5 As shown in Figure A, compared with the control group, the EHP copy number in shrimp was significantly reduced after feeding the diet containing Evodia rutaecarpa ethanol extract for 20 and 30 days. Figure 5 As shown in Figure B, the weight of EHP-infected shrimp increased by 12% after being fed with the diet containing the alcohol extract of Evodia rutaecarpa for 30 days compared with the control group. The above results indicate that these Chinese medicinal alcohol extracts have the potential to be used in the treatment of EHP-infected shrimp.

[0031] Specific Example 3: Analysis of the anti-EHP activity of the compound of ethanol extract of Sophora alopecuroides, ethanol extract of Fraxinus frasii and ethanol extract of Evodia rutaecarpa.

[0032] First, the compound formulas were prepared according to the weight ratio: Compound A (ethanol extract of Sophora alopecursii: alcohol extract of Fraxinus chinensis: alcohol extract of Evodia rutaecarpa = 10:60:30), Compound B (ethanol extract of Sophora alopecursii: alcohol extract of Fraxinus chinensis: alcohol extract of Evodia rutaecarpa = 20:55:25) and Compound C (ethanol extract of Sophora alopecursii: alcohol extract of Fraxinus chinensis: alcohol extract of Evodia rutaecarpa = 15:75:10). Healthy shrimps were fed with fresh hepatopancreas of EHP-infected shrimps at a feeding amount of 2 g / 10 shrimps and divided into two groups, with 30 shrimps in each group. After 7 days, they were fed with commercial feed containing 0.5wt% Compound A, 0.5wt% Compound B and 0.5wt% Compound C, respectively, and the control group was fed with commercial feed containing 0.5wt% DMSO, three times a day, 1 g each time. On the 10th, 20th and 30th days after feeding, the hepatopancreas of shrimp was taken to detect the EHP copy number, and the body weight of shrimp in each group was weighed.

[0033] The results are as follows Figure 6 Middle A, Figure 7 Middle A and Figure 8 As shown in Figure A, after feeding the diets containing Compound A, Compound B, and Compound C for 10, 20, and 30 days, the EHP copy numbers in shrimp were significantly reduced. Figure 6 Middle B, Figure 7 Medium B and Figure 8 As shown in Figure B, in terms of body weight, after being fed with feeds containing Compound A, Compound B, and Compound C for 30 days, the weight of EHP-infected shrimp increased by 22%, 18%, and 16%, respectively, and there was a significant difference between the shrimp fed with feed containing Compound A and the control group. The above results show that Compound A has the best therapeutic effect on EHP-infected shrimp.

[0034] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A Chinese medicinal compound for resisting shrimp hepatoenteric cytozoon, characterized in that The invention is composed of the following raw materials and their weight proportions: 10-20 parts of alcohol extract of sophora alopecuroides, 50-75 parts of alcohol extract of fraxinus chinensis and 10-30 parts of alcohol extract of evodia rutaecarpa.

2. The Chinese medicinal compound for resisting shrimp hepatoenteric cytozoon according to claim 1, characterized in that The invention is composed of the following raw materials and their weight proportions: 10 parts of alcohol extract of Sophora alopecuroides, 60 parts of alcohol extract of Fraxinus frassinicola and 30 parts of alcohol extract of Evodia rutaecarpa.

3. The Chinese medicinal compound for resisting shrimp hepatoenteric cytozoon according to claim 1, characterized in that The invention is composed of the following raw materials and their weight proportions: 20 parts of sophora alopecuroides extract, 55 parts of fraxinus ethanol extract and 25 parts of evodia rutaecarpa ethanol extract.

4. The Chinese medicinal compound for resisting shrimp hepatoenteric cytozoon according to claim 1, characterized in that The invention is composed of the following raw materials and their weight proportions: 15 parts of sophora alopecuroides extract, 75 parts of fraxinus ethanol extract and 10 parts of evodia rutaecarpa ethanol extract.

5. A Chinese medicinal compound for resisting shrimp hepatoenteric cytozoon according to any one of claims 1 to 4, characterized in that The preparation methods of the alcohol extract of bitter bean, the alcohol extract of frasin and the alcohol extract of Evodia rutaecarpa are as follows: bitter bean, frasin and Evodia rutaecarpa are dried and crushed, 50 g of each are weighed and added with 300 mL of anhydrous ethanol after being sieved through a 100-mesh sieve, and the mixture is placed in an ultrasonic cleaner for ultrasonic dissolution for 10 min, and then placed in a 60°C water bath for heating for 30 min, the above steps are repeated 3 times, 150 mL of anhydrous ethanol is added each time, the anhydrous ethanol extract is concentrated by rotary evaporation, and finally placed in an oven for drying to obtain the alcohol extract of bitter bean, the alcohol extract of frasin and the alcohol extract of Evodia rutaecarpa, respectively.

6. A method for preparing the anti-shrimp hepatocellular carcinoma Chinese medicinal compound according to claim 5, characterized in that The following steps are involved: (1) Weigh the dried and crushed bitter bean, qinpi and rutaecarpa, and use anhydrous ethanol to dissolve and extract to obtain ethanol extracts; (2) Weigh 10-20 parts of the alcohol extract of Sophora alopecuroides, 50-75 parts of the alcohol extract of Fragrant Cortex and 10-30 parts of the alcohol extract of Evodia rutaecarpa by weight, and mix them evenly to obtain the anti-EHP Chinese medicine compound.

7. Use of the anti-shrimp hepatoenteric cytozoon Chinese medicinal compound according to claim 1 in the preparation of shrimp hepatoenteric cytozoon inhibitors.

8. Use of the alcohol extract of Sophora alopecuroides according to claim 1 in the preparation of shrimp hepato-intestinal cytozoon inhibitors, characterized in that: The amount of the alcohol extract of Sophora alopecuroides added to the shrimp breeding water is 10-40 mg / L.

9. Use of the alcohol extract of Cortex Fraxini according to claim 1 in the preparation of shrimp hepato-enteric cytozoon inhibitors, characterized in that: The amount of the alcohol extract of the Fraxinus fraxinus added to the shrimp breeding water is 10-80 mg / L.

10. Use of the alcohol extract of Evodia rutaecarpa according to claim 1 in the preparation of shrimp hepato-enteric cytozoon inhibitors, characterized in that: The amount of the Evodia rutaecarpa alcohol extract added to the shrimp breeding water is 10-80 mg / L.

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