A traditional Chinese medicine compound for resisting shrimp hepatoenteric cysts, and its preparation method and application

Abstract

The invention discloses a traditional Chinese medicine compound for resisting shrimp hepatoenteric cytozoon, a preparation method and application thereof. The traditional Chinese medicine compound is characterized by being composed of the following raw materials and their weight proportions: 10-20 parts of a sophora alopecuroides ethanol extract, 50-75 parts of a frasinic cortex ethanol extract and 10-30 parts of an rutaecarpa ethanol extract. The preparation method comprises the following steps: weighing dried and crushed sophora alopecuroides, frasinic cortex and rutaecarpa, respectively dissolving and extracting the extracts using anhydrous ethanol to obtain the extracts; weighing the traditional Chinese medicine extracts according to weight proportions, and mixing the extracts to obtain the traditional Chinese medicine compound for resisting shrimp hepatoenteric cytozoon. The invention also provides application of the traditional Chinese medicine compound for resisting shrimp hepatoenteric cytozoon, the sophora alopecuroides ethanol extract, the frasinic cortex ethanol extract and the rutaecarpa ethanol extract in preparing a shrimp hepatoenteric cytozoon inhibitor. The traditional Chinese medicine compound has the advantages of inhibiting the proliferation of shrimp hepatoenteric cytozoon in shrimp, increasing the weight gain rate of shrimp, and effectively reducing the economic losses caused by shrimp hepatoenteric cytozoon outbreaks to shrimp farming. The compound has low cost and a simple application method.

Description

Technical Field

[0001] The present 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 (Epidemiaceous Hepatocellular Parkinson's Disease), an obligate intracellular parasite, causes enterocytosis in shrimp, a disease classified as a Category II animal disease by my country's Ministry of Agriculture. EHP reduces shrimp feeding capacity, slows growth, or even halts growth, leading to feed depletion and a high risk of secondary bacterial infections. Its multiple vectors, wide distribution, and high transmission rate pose a serious threat to the sustainable development of aquaculture. The primary control strategy for aquatic parasitic diseases is medication. However, existing anti-insecticides such as pyrethroids, trichlorfon, and mebendazole not only have limited efficacy, but also face food and environmental safety concerns, further limiting their application in aquaculture. Traditional Chinese medicine (TCM) has attracted considerable attention for its natural safety, minimal toxicity, low residue, and resistance to drug resistance, making it a promising candidate for aquatic animal disease control. Studies have shown that the TCM herbs Sophora alopecuroides, Fraxinus chinensis, and Evodia rutaecarpa possess significant antibacterial, insecticidal, antioxidant, and immunomodulatory properties. 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-prawn hepatoenterocyte activity and the anti-prawn hepatoenterocyte activity, as well as 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 parts: 10-20 parts of sophora alopecuroides ethanol extract, 55-75 parts of fraxinus 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 parts: 10 parts of Sophora alopecuroides extract, 60 parts of Fraxinus fraxinus 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 Fraxinus fraxinus 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 Fraxinus fraxinus ethanol extract and 10 parts of Evodia rutaecarpa ethanol extract.

[0008] Furthermore, the preparation method of the alcohol extract of Sophora alopecuroides, the alcohol extract of Fraxinus frass and the alcohol extract of Evodia rutaecarpa is as follows: the Sophora alopecuroides, the Fraxinus frass and the 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, and the mixture is placed in an ultrasonic cleaner for ultrasonic dissolution for 10 min, and then heated in a 60°C water bath for 30 min, and the above steps are repeated 3 times, adding 150 mL of anhydrous ethanol each time, and the anhydrous ethanol extract is concentrated by rotary evaporation, and finally placed in an oven for drying to obtain the alcohol extract of Sophora alopecuroides, the alcohol extract of Fraxinus frass and the alcohol extract of Evodia rutaecarpa, respectively.

[0009] The present invention also provides a method for preparing the above-mentioned traditional Chinese medicine compound for resisting shrimp hepatoenteric cytozoon, comprising the following steps:

[0010] (1) Weigh the dried and crushed bitter beans, qinpi and rutaecarpa, and dissolve them in anhydrous ethanol to obtain ethanol extracts;

[0011] (2) Weigh 10-20 parts of the alcohol extract of Sophora alopecuroides, 50-75 parts of the alcohol extract of Fraxinus fraxinus, 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.

[0012] 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.

[0013] The present invention also provides the use of the alcohol extract of Sophora alopecuroides in the preparation of shrimp hepato-intestinal cytozoon inhibitors, wherein the alcohol extract of Sophora alopecuroides is added in an amount of 10-40 mg / L to shrimp aquaculture water.

[0014] The present invention also provides the use of the above-mentioned Fraxinus fraxinus alcohol extract in the preparation of shrimp hepatoenteric cytozoon inhibitors, wherein the addition amount of the Fraxinus fraxinus alcohol extract to shrimp aquaculture water is 10-80 mg / L.

[0015] The present invention also provides the use of the above-mentioned Evodia rutaecarpa ethanol extract in the preparation of shrimp hepato-enteric cytozoon inhibitors, wherein the amount of the Evodia rutaecarpa ethanol extract added to shrimp aquaculture water is 10-80 mg / L.

[0016] Compared with the existing technology, the present invention has the advantages of providing a traditional Chinese medicine compound for preventing hepatocystis in shrimp, its preparation method, and its use. The compound comprises drying and pulverizing bitter beans, frasin bark, and Evodia rutaecarpa, extracting the active ingredients with anhydrous ethanol, and then weighing the ethanol extract in proportion and mixing it to obtain the anti-EHP traditional Chinese medicine compound. The compound can effectively block EHP outbreaks. The anti-EHP effect of the traditional Chinese medicine compound is more significant than the use of the ethanol extracts of bitter beans, frasin bark, and Evodia rutaecarpa alone. Furthermore, the compound is simple to prepare and use, is natural and pollution-free, and can effectively reduce the economic losses caused by EHP outbreaks to shrimp farming. The compound is low-cost, simple to use, and easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of Chinese herbal medicine ethanol extracts on the survival rate of shrimp, where A is the ethanol extract of Sophora alopecuroides, B is the ethanol extract of Fraxinus chinensis, and C is the ethanol extract of Evodia rutaecarpa.

[0018] Figure 2 To analyze the anti-EHP activity of Chinese herbal medicine ethanol extracts;

[0019] Figure 3 The results show that the alcohol extract of Sophora alopecuroides treats EHP-infected shrimps, where A shows the effect of the alcohol extract of Sophora alopecuroides on the number of EHP copies, and B shows the effect of the alcohol extract of Sophora alopecuroides on the body weight of EHP-infected shrimps.

[0020] Figure 4 The treatment of EHP-infected shrimp with the alcohol extract of Fraxinus fraxinus, where A is the effect of the alcohol extract of Fraxinus fraxinus on the EHP copy number, and B is the effect of the alcohol extract of Fraxinus fraxinus on the body weight of EHP-infected shrimp;

[0021] 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 EHP copy number, and B is the effect of Evodia rutaecarpa ethanol extract on the body weight of shrimp infected with EHP;

[0022] Figure 6 is the treatment of compound A on EHP-infected shrimp, 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 EHP-infected shrimp;

[0023] Figure 7 is the treatment of compound B on EHP-infected shrimp, 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 EHP-infected shrimp;

[0024] Figure 8 Figure 3 is the treatment of compound C on EHP-infected shrimp, 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 EHP-infected shrimp. DETAILED DESCRIPTION

[0025] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0026] To illustrate the essence of the present invention, the anti-EHP activity of ethanol extracts of Sophora alopecuroides, Fraxinus frassinicola, and Evodia rutaecarpa, as well as their combination, was analyzed using Litopenaeus vannamei as the experimental subject. Sophora alopecuroides, Fraxinus frassinicola, Evodia rutaecarpa, dimethyl sulfoxide (DMSO), and anhydrous ethanol were all provided by Shanghai Aladdin Biochemical Technology Co., Ltd., while EHP and Litopenaeus vannamei were provided by Zhejiang Marine Aquaculture Research Institute.

[0027] Preparation of mother liquors of alcohol extracts of Sophora alopecuroides, Fraxinus cortex, and Evodia rutaecarpa: Dry and pulverize Sophora alopecuroides, Fraxinus cortex, and Evodia rutaecarpa. After sieving through a 100-mesh sieve, weigh 50 g of each and add 300 mL of anhydrous ethanol. Place in an ultrasonic cleaner and sonicate for 10 min. Then, heat in a 60°C water bath for 30 min. Repeat this step three times, adding 150 mL of anhydrous ethanol each time. The anhydrous ethanol extracts were concentrated by rotary evaporation and oven-dried to obtain alcohol extracts of Sophora alopecuroides, Fraxinus cortex, and Evodia rutaecarpa. Each of the alcohol extracts was fully dissolved in DMSO to a final concentration of 50,000 mg / L. The stock liquors were stored at 4°C until use.

[0028] EHP copy number detection: Shrimp DNA was extracted using a marine animal tissue genomic DNA rapid extraction kit (Tiangen). DNA concentration (adjusted to a final concentration of 100 ng / μL) and purity were detected using an ultra-micro spectrophotometer, followed by qPCR amplification. 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'. The qPCR amplification reaction system is as follows: 7.5 μL of 2 × Es Taq MasterMix, 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; 35 cycles of 95°C for 30 s, 55.5°C for 30 s, and 72°C for 30 s; and 72°C for 10 min. The pMD19T-ENF 185 The standard curve made from the standard product was used to convert the qPCR results to obtain the EHP copy number.

[0029] Specific Example 1: Toxicity experiments on ethanol extracts of Sophora alopecuroides, Fraxinus fraxinus and Evodia rutaecarpa.

[0030] Healthy shrimp were divided into 5 groups, with 15 shrimp in each group. The experimental setting was immersion method. The concentration of the alcohol extract of Sophora alopecuroides in the aquaculture water was 0, 10, 20, 40, 80 and 160 mg / L respectively. The control group was a DMSO concentration of 0.02% in the aquaculture water. The survival rate of shrimp was calculated after 72 hours, and the safe concentration of the alcohol extract of Sophora alopecuroides was determined based on the shrimp survival rate. 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.

[0031] Similarly, the experimental setting of the concentration of the alcohol extract of Fraxinus chinensis in the aquaculture water was 0, 10, 20, 40, 80 and 160 mg / L, and the control group was a DMSO concentration of 0.02% in the aquaculture water. The survival rate of the shrimp was calculated after 72 hours. The results are as follows Figure 1 As shown in Figure 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.

[0032] The same experimental setting was used, with the concentrations of Evodia rutaecarpa ethanol extract in the aquaculture water being 0, 10, 20, 40, 80, and 160 mg / L, respectively. The control group was a DMSO concentration of 0.02% in the aquaculture water. The survival rate of the shrimp was calculated after 72 hours. The results are as follows: Figure 1 As shown in Figure 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.

[0033] Specific Example 2: Analysis of the anti-EHP activity of the ethanol extracts of Sophora alopecuroides, Fraxinus fraxinus and Evodia rutaecarpa.

[0034] 1. Preliminary analysis of the anti-EHP activity of ethanol extracts of Sophora alopecuroides, Fraxinus fraxinus, and Evodia rutaecarpa: The experiment was set up with three groups of aquaculture water supplemented with 40 mg / L of ethanol extract of Sophora alopecuroides, 80 mg / L of ethanol extract of Fraxinus fraxinus, and 80 mg / L of ethanol extract of Evodia rutaecarpa. The control group was fed with aquaculture water containing 0.02 wt% 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 rate was 2 g / 10 shrimp, with 10 shrimp per group. On day 7 of infection, samples were collected and analyzed for EHP copy number using qPCR.

[0035] The results are as follows Figure 2 As shown in the results, compared with the control group, the ethanol extracts of Sophora alopecuroides, Fraxinus fraxinus and Evodia rutaecarpa could significantly reduce the EHP copy number in shrimp, with the inhibition rates of EHP copy number being 50%, 47% and 58%, respectively, indicating that the above-mentioned ethanol extracts all have a certain effect of inhibiting the proliferation of EHP.

[0036] 2. Analysis of the anti-EHP activity of ethanol extracts of Sophora alopecuroides, Fraxinus fraxinus, and Evodia rutaecarpa: Healthy shrimp were fed fresh hepatopancreas from EHP-infected shrimp at a dose of 2 g / 10 shrimp. The shrimp were then divided into two groups, each containing 30 shrimp. Seven days later, the shrimp were fed a commercial feed containing 0.5 wt% ethanol extract of Sophora alopecuroides, 0.5 wt% ethanol extract of Fraxinus fraxinus, and 0.5 wt% ethanol extract of Evodia rutaecarpa. The control group was fed a commercial feed containing 0.5 wt% DMSO, three times daily, at a dose of 1 g each. On days 10, 20, and 30 after feeding, the hepatopancreas of the shrimp were collected to measure the EHP copy number, and the shrimp in each group were weighed.

[0037] 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 Figure B, the body weight of shrimp infected with EHP increased by 11% after 30 days of feeding compared with the control group, but there was no significant difference.

[0038] 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 fraxinus for 10, 20 and 30 days. Figure 4 As shown in Figure B, compared with the control group, the weight of EHP-infected shrimp increased by 14% after being fed with the diet containing the alcohol extract of Fraxinus fraxini for 30 days.

[0039] 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, EHP-infected shrimp fed a diet containing the ethanol extract of Evodia rutaecarpa gained 12% more weight after 30 days of feeding compared to the control group. These results suggest that these Chinese herbal extracts have the potential to treat EHP-infected shrimp.

[0040] Specific Example 3: Analysis of the anti-EHP activity of the compound of the ethanol extract of Sophora alopecuroides, the ethanol extract of Fraxinus chinensis and the ethanol extract of Evodia rutaecarpa.

[0041] First, compound formulas were prepared according to the following weight ratios: Compound A (ethanol extract of Sophora alopecuroides: ethanol extract of Fraxinus officinalis: ethanol extract of Evodia rutaecarpa = 10:60:30), Compound B (ethanol extract of Sophora alopecuroides: ethanol extract of Fraxinus officinalis: ethanol extract of Evodia rutaecarpa = 20:55:25), and Compound C (ethanol extract of Sophora alopecuroides: ethanol extract of Fraxinus officinalis: ethanol extract of Evodia rutaecarpa = 15:75:10). Healthy shrimp were fed fresh hepatopancreas from EHP-infected shrimp at a dose of 2 g / 10 shrimp and divided into two groups, each containing 30 shrimp. After 7 days, the shrimp were fed a commercial feed containing 0.5 wt% Compound A, 0.5 wt% Compound B, and 0.5 wt% Compound C, respectively. The control group was fed a commercial feed containing 0.5 wt% DMSO, three times daily, 1 g each time. On the 10th, 20th and 30th day after feeding, the hepatopancreas of shrimp was collected to detect the EHP copy number, and the body weight of shrimp in each group was weighed.

[0042] The results are as follows Figure 6 Middle A, Figure 7 China A and Figure 8 As shown in Figure A, after feeding the diet containing Compound A, Compound B and Compound C for 10, 20 and 30 days, the EHP copy number in shrimp was significantly reduced. Figure 6 Middle B, Figure 7 Middle B and Figure 8 As shown in Figure B, after 30 days of feeding with compound A, compound B, and compound C, EHP-infected shrimp gained 22%, 18%, and 16% more weight, respectively. The shrimp fed with compound A showed a significant difference compared to the control group. These results indicate that compound A is the most effective treatment for EHP-infected shrimp.

[0043] 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 persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine compound for resisting shrimp hepatoenteric cysts, characterized in that The invention comprises the following raw materials and their weight parts: 10-20 parts of alopecuroides alcohol extract, 50-75 parts of frasin cortex alcohol extract and 10-30 parts of evodia rutaecarpa alcohol extract, wherein the preparation method of the alopecuroides alcohol extract, the frasin cortex alcohol extract and the evodia rutaecarpa alcohol extract is as follows: taking the alopecuroides, the frasin cortex and the evodia rutaecarpa ethanol extract, drying and grinding them, sieving them through a 100-mesh sieve, weighing 50 g of each, adding 300 mL of anhydrous ethanol, placing them in an ultrasonic cleaner for ultrasonic dissolution for 10 minutes, and then heating them in a 60°C water bath for 30 minutes, repeating the above steps three times, adding 150 mL of anhydrous ethanol each time, and concentrating the anhydrous ethanol extract by rotary evaporation. Finally, the extracts are dried in an oven to obtain the alopecuroides alcohol extract, the frasin cortex alcohol extract and the evodia rutaecarpa ethanol extract, respectively.

2. The Chinese medicinal compound for resisting shrimp hepato-enteric cytozoon according to claim 1, characterized in that The preparation comprises the following raw materials and their weight proportions: 10 parts of Sophora alopecuroides ethanol extract, 60 parts of Fraxinus fraxinus ethanol extract and 30 parts of Evodia rutaecarpa ethanol extract.

3. The Chinese medicinal compound for resisting shrimp hepato-enteric cytozoon according to claim 1, characterized in that The preparation comprises the following raw materials and their weight proportions: 20 parts of Sophora alopecuroides extract, 55 parts of Fraxinus fraxinus ethanol extract and 25 parts of Evodia rutaecarpa ethanol extract.

4. The Chinese medicinal compound for resisting shrimp hepatocellular carcinoma according to claim 1, characterized in that The preparation comprises the following raw materials and their weight proportions: 15 parts of Sophora alopecuroides extract, 75 parts of Fraxinus fraxinus ethanol extract and 10 parts of Evodia rutaecarpa ethanol extract.

5. A method for preparing the anti-shrimp hepato-enteric cytozoon Chinese medicinal compound according to any one of claims 1 to 4, characterized in that The following steps are involved: (1) Weigh the dried and crushed bitter beans, qinpi and rutaecarpa, and dissolve them in anhydrous ethanol 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 a traditional Chinese medicine compound for resisting shrimp hepatoenteric cytozoon.

6. Use of the traditional Chinese medicine compound against shrimp hepatoenteric cytozoa according to claim 1 in the preparation of shrimp hepatoenteric cytozoa inhibitors.

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