A Chinese medicinal composition for preventing and treating mitten crab (Eriocheir sinensis) infection, and its preparation method and application

Abstract

The present invention provides a Chinese medicine composition for preventing and treating "milk disease" of Chinese mitten crab, and a preparation method and application thereof, and the present invention belongs to the technical field of Chinese medicine for aquatic animals. The Chinese medicine composition provided by the present invention comprises the following raw materials in parts by weight: 1 to 10 parts of coptis root, 1 to 10 parts of sappan wood, 1 to 10 parts of pomegranate peel, 1 to 10 parts of cloves, 1 to 10 parts of scutellaria and 1 to 10 parts of schisandra chinensis. The Chinese medicine composition provided by the present invention is for the prevention and treatment of "milk disease" of Chinese mitten crab caused by Metschnikowia bicuspidatum, and provides an efficient, safe and practical method, with an inhibition rate of more than 99.67%, low cost, simple preparation process, high raw material utilization rate, easy industrialization, and good development value and market prospects.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine for aquatic animals, and in particular to a traditional Chinese medicine composition for preventing and treating the infection of Eriocheir sinensis, and a preparation method and application thereof. Background Art

[0002] Chinese mitten crab is one of the most important crustaceans in aquaculture and is very popular and loved by consumers. However, with the continuous expansion of aquaculture scale, the increase of aquaculture density and the deterioration of aquaculture environment conditions, a variety of aquaculture diseases have occurred, which seriously restricted the healthy development of crab aquaculture. The outbreak of "milk disease" has caused the mortality rate of Chinese mitten crab to exceed 20%, resulting in huge losses for farmers. The main clinical symptoms of the disease are slow reaction, swollen joints, loss of walking legs, opaque or whitish muscles. Pathological examination found that the muscle fiber structure of the diseased crab was loose, the horizontal stripes disappeared, and some areas were dissolved and liquefied. A large amount of white milky liquid was seen when the carapace was opened. The muscles of the whole body were edematous and not full, which eventually led to tissue or organ failure and crab death. The researchers isolated a strain of Metschnikowia bicuspidata from the diseased crab. The artificial infection experiment found that the strain was pathogenic to healthy Chinese mitten crabs and showed the same symptoms as naturally diseased crabs. It was determined that Metschnikowia bicuspidata was the source of the "milk disease" of Chinese mitten crabs in northern China. Therefore, developing effective fungicides or inhibitors to inhibit the reproduction and growth of this strain is the most effective measure to reduce the incidence of "milk disease" in river crabs, and it is also the top priority for disease prevention and control in river crab farming.

[0003] As natural substances, Chinese herbal medicines are widely available, inexpensive, and contain a variety of biologically effective ingredients. They have good antibacterial properties, low toxicity, and do not pollute the environment. While inhibiting the reproduction of pathogens, they ensure the quality and safety of aquatic products. In the current context of "reducing and banning antibiotics", it is particularly important to develop antibacterial Chinese herbal medicine compositions to replace the use of antibiotics and chemical drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a Chinese medicinal composition which is efficient, economical, safe and easy to industrialize for preventing and treating "milk disease" of river crabs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an application of a traditional Chinese medicine in preparing a medicine for preventing and treating "milk disease" of Chinese mitten crab. The traditional Chinese medicine is one of coptis chinensis, sappan wood, pomegranate peel, clove, scutellaria baicalensis and schisandra chinensis.

[0007] The present invention also provides a traditional Chinese medicine composition using the above traditional Chinese medicine as raw materials, wherein the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 1 to 10 parts of coptis chinensis, 1 to 10 parts of sappan wood, 1 to 10 parts of pomegranate peel, 1 to 10 parts of cloves, 1 to 10 parts of scutellaria baicalensis and 1 to 10 parts of schisandra chinensis.

[0008] Preferably, the Chinese medicine composition comprises the following raw materials in parts by weight: 5 parts of coptis chinensis, 5 parts of sappan wood, 4 parts of pomegranate peel, 5 parts of cloves, 5 parts of scutellaria baicalensis and 5 parts of schisandra chinensis.

[0009] The present invention also provides a method for preparing the above-mentioned Chinese medicine composition, comprising the following steps:

[0010] (1) soaking the raw drug in water, boiling, and separating the solid from the liquid to obtain drug residue and the first drug solution;

[0011] (2) taking the medicinal residue and continuing to boil it, separating the solid from the liquid, and obtaining a second medicinal solution;

[0012] (3) combining the first medicinal solution and the second medicinal solution, concentrating them, and obtaining the traditional Chinese medicine composition.

[0013] The mass volume ratio of the raw material drug and water is 1g:10-20mL.

[0014] Preferably, the decocting time in step (1) and step (2) is 30 to 40 minutes.

[0015] Preferably, the crude drug content of the traditional Chinese medicine composition is 0.5-1 g / ml.

[0016] Preferably, the concentrated medicinal solution is sterilized at high temperature and refrigerated for later use.

[0017] Preferably, the sterilization temperature is 100-121° C. and the time is 15-20 min.

[0018] The present invention also provides an application of the traditional Chinese medicine composition obtained by the above preparation method in preparing medicine for preventing and treating "milk disease" of Chinese mitten crab.

[0019] The Chinese medicine composition for preventing and treating the pathogenic bacteria of "milk disease" provided by the present invention is the first to use a combination of Chinese medicines to prevent and treat the pathogen. Compared with the chemical drugs of dithiocyanomethane, benzalkonium bromide and glutaraldehyde used for prevention and treatment in CN202210194798.2, the positive effects thereof are:

[0020] (1) The diameter of the inhibition zone of Coptis chinensis against Saccharomyces cerevisiae reached 40.5 mm, which was higher than the diameter of the inhibition zone of the combination of dithiocyanate, benzalkonium bromide, and glutaraldehyde (35.79 mm), indicating that the Chinese medicine preparation has a strong antibacterial ability.

[0021] (2) The Chinese medicine combination selected by the present invention, namely 5 parts of coptis root, 5 parts of sappan wood, 4 parts of pomegranate peel, 5 parts of clove, 5 parts of scutellaria baicalensis, and 5 parts of schisandra chinensis, has an antibacterial rate of 99.67% against Metschnikowia bicuspidatum, which is significantly higher than that of a single Chinese medicine at the same concentration.

[0022] (3) The present invention is a traditional Chinese medicine product, which has low toxicity and side effects on farmed organisms, no residue, and no environmental pollution, and meets the requirements for sustainable development of the aquaculture industry.

[0023] (4) The resource of the present invention is easy to obtain, the cost is low, the traditional process is used for boiling, the operation is simple, the raw material utilization rate is high, and it is suitable for large-scale production.

[0024] (5) The present invention provides a better method for preventing and treating aquatic animal diseases caused by Metschnikowia bicuspidatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The diameters of the inhibition zones of different Chinese medicinal solutions against Saccharomyces cerevisiae were determined by the Oxford cup method in Example 1;

[0026] Figure 2 The inhibition zones of different Chinese medicine extracts measured by the Oxford cup method in Example 1;

[0027] Figure 3 The 96-well plate method in Example 2 was used to determine the antibacterial rate of different concentrations of Chinese medicine liquid on Saccharomyces cerevisiae;

[0028] Figure 4 The inhibition rate of the optimal Chinese medicine compound combination and the single Chinese medicine liquid against Saccharomyces cerevisiae in Example 4 is shown. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] 1. Preparation of Chinese medicine solution

[0032] Take 30g of each of the Chinese herbal medicines of Coptis chinensis, Sappan wood, pomegranate peel, clove, Scutellaria baicalensis, and Schisandra chinensis, add 300mL of distilled water to soak at room temperature (25℃) for 60min, then heat to boiling, decoct for 30min under boiling condition, pour out the liquid, add 300mL of water to the residue, and decoct for 30min. Combine the two liquids and filter, evaporate and concentrate the filtrate to 30mL, so that the crude drug content of the liquid is 1g / mL. After sterilization at 121℃ for 20min, store the prepared liquid in a refrigerator at 4℃ for later use.

[0033] 2. Antibacterial activity test

[0034] In vitro antibacterial test---Oxford cup method

[0035] The activated yeast solution (concentration of 10 8 cfu / ml) was evenly spread on a culture dish containing LB medium. An Oxford cup was placed on the medium, and 100 μL of the 6 traditional Chinese medicine solutions prepared above were injected into the Oxford cup. An equal volume of sterilized water was added as a control. The mixture was allowed to stand at room temperature for 3 hours, and then placed in a 32±2℃ incubator for 48 hours. The size of the inhibition zone was measured with a vernier caliper.

[0036] For specific results, see Figure 1 , Figure 2 It can be seen that the six Chinese medicines have good antibacterial effects on Saccharomyces cerevisiae, among which Coptis chinensis has the strongest antibacterial effect, with an inhibition zone diameter of 40.5 mm, followed by Scutellaria baicalensis and pomegranate peel, which are 30.5 mm and 29.75 mm respectively, while Sappan wood and cloves are 21 mm, and Schisandra chinensis is 16 mm.

[0037] Example 2

[0038] In vitro antibacterial test-----96-well plate method

[0039] The Chinese medicine solution was prepared according to the method of Example 1. The antibacterial effects of different concentrations of 6 Chinese medicines with good antibacterial properties, namely, coptis root, sappan wood, pomegranate peel, clove, scutellaria baicalensis, and schisandra chinensis, were determined using a 96-well plate method to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of different Chinese medicines.

[0040] Add 10 μL of yeast culture medium, 10 μL of Chinese medicine solution, and 180 μL of sterilized LB culture medium to a 96-well plate. Use 10 μL of sterile water instead of Chinese medicine solution as a control. Repeat 3 times for each treatment. Place the plate in a 30°C incubator and culture for 24 hours. Measure OD with a microplate reader. 590nm The antibacterial rate of traditional Chinese medicine was calculated according to formula (1).

[0041] Antibacterial rate (%) formula (1)

[0042]

[0043] In formula (1), ΔOD1 represents the OD of pathogens in the treatment group. 590nm Increment; ΔOD0 represents the OD of pathogens in the control group 590nm Increment.

[0044] Set different concentration gradients of 400, 200, 100, 50, 25, 12.5, 6.2, 3.1, and 1 mg / mL, use sterile water as the control, add different concentrations of drug solution to the 96-well plate, culture at 30°C in a constant temperature incubator for 24 hours, observe the growth of the bacterial solution, and record the results. The lowest drug concentration that completely inhibits the growth of pathogens in the well is the MIC.

[0045] The culture fluid of the MIC concentration point and the two treatments before and after the MIC concentration point (3 wells for each treatment) on the 96-well plate was aspirated and coated on the LB medium plate. The plate was cultured at 30°C and the colonies were counted. The drug concentration with less than 5 colonies was the MBC.

[0046] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 18 g / L agar for solid medium.

[0047] The antibacterial effects of different concentrations Figure 3 ,Depend on Figure 3 We can see that Coptis chinensis has the highest antibacterial rate. At a concentration of 200-400 mg / mL, the pathogenic yeast Mycorrhiza uralensis basically does not grow, and the antibacterial rate reaches 100%. The antibacterial rate of 25 mg / mL treatment also reaches more than 50%. The second is Schisandra chinensis. At a concentration of 100-400 mg / mL, the antibacterial rate exceeds 97%, and the antibacterial rate of 50 mg / mL treatment also reaches more than 50%. Cloves and Scutellaria baicalensis also have an antibacterial rate of more than 50% at a concentration of 100 mg / mL. The antibacterial rates of pomegranate peel and Sappan wood reach more than 50% at 200 mg / mL.

[0048] The results of the minimum inhibitory concentration and minimum bactericidal concentration of different drugs are shown in Table 1

[0049] Table 1 Minimum inhibitory concentration and minimum bactericidal concentration of different Chinese herbal medicine extracts against Saccharomyces cerevisiae

[0050]

[0051]

[0052] Example 3

[0053] Screening of compound Chinese medicine

[0054] Compound screening was carried out based on the single Chinese medicines screened in the early stage. Each Chinese medicine was one factor, and each factor had five concentration levels (Table 2). The antibacterial rate was used as an important indicator for screening compound prescriptions. The design was carried out according to the 6-factor, 5-level orthogonal design, and different compounding ratios were set to screen out the optimal compound combination.

[0055] Table 2 Levels of orthogonal design factors for different traditional Chinese medicines

[0056]

[0057] Table 3 Orthogonal design test results of different traditional Chinese medicines

[0058]

[0059]

[0060] The preferred ratio is 5 parts of coptis chinensis, 5 parts of sappan wood, 4 parts of pomegranate peel, 5 parts of cloves, 5 parts of scutellaria baicalensis, and 5 parts of schisandra chinensis, that is, 10 mg / mL of coptis chinensis, 10 mg / mL of sappan wood, 8 mg / mL of pomegranate peel, 10 mg / mL of cloves, 10 mg / mL of scutellaria baicalensis, and 10 mg / mL of schisandra chinensis. The total concentration is 58 mg / mL, and the antibacterial rate against milk disease can reach 99.67%±0.14%.

[0061] Example 4

[0062] According to the preparation method of Example 1, each Chinese medicine concentrate was prepared. The screened composition, i.e., 10 mg / mL of Coptis chinensis, 10 mg / mL of Sappan wood, 8 mg / mL of pomegranate peel, 10 mg / mL of cloves, 10 mg / mL of scutellaria baicalensis, and 10 mg / mL of Schisandra chinensis, with a total concentration of 58 mg / mL, was compounded, and the concentrates of the six Chinese medicines were diluted to 58 mg / mL. The antibacterial rates of the single compound were compared by the 96-well plate method. The results showed that at the same concentration, the antibacterial rate of the Chinese medicine compound combination reached more than 99.67%, which was significantly higher than the antibacterial rate of the single Chinese medicine. The results are as follows: Figure 4 .

[0063] As can be seen from the above embodiments, the present invention provides a Chinese medicine composition for preventing and treating the pathogenic bacteria of "milk disease", a preparation method and application thereof. The Chinese medicine composition of the present invention has a high antibacterial rate, is easy to obtain resources, has a low cost, is simple to operate by using traditional technology, has a high raw material utilization rate, and is suitable for large-scale production.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicinal composition for preventing and treating infection by Metschnikowia bicuspidatum in Chinese mitten crab, characterized in that: The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 5 parts of coptis root, 5 parts of sappan wood, 4 parts of pomegranate peel, 5 parts of cloves, 5 parts of scutellaria baicalensis and 5 parts of schisandra chinensis.

2. The method for preparing the Chinese medicine composition according to claim 1, characterized in that: The steps include: (1) soaking the raw material in water, boiling, and separating the solid from the liquid to obtain a drug residue and a first drug solution; (2) taking the medicinal residue and continuing to boil it, separating the solid and the liquid, and obtaining a second medicinal solution; (3) combining the first medicinal solution and the second medicinal solution, concentrating them, and obtaining the traditional Chinese medicine composition; The crude drug content of the traditional Chinese medicine composition is 0.5-1 g / ml.

3. The method for preparing the Chinese medicine composition according to claim 2, characterized in that: The mass volume ratio of the raw material drug and water is 1g:10~20ml.

4. The method for preparing the Chinese medicine composition according to claim 3, characterized in that: The soaking time is 60 to 120 minutes; the decoction time in step (1) and step (2) is 30 to 40 minutes.

5. Use of the Chinese medicine composition according to claim 1 or the Chinese medicine composition obtained by the preparation method according to any one of claims 2 to 4 in the preparation of a medicament for preventing and treating infection by Saccharomyces cerevisiae in Eriocheir sinensis.

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