A traditional Chinese medicine composition for preventing and treating sea cucumber rot syndrome, preparation method and application thereof

Abstract

The present invention belongs to the field of traditional Chinese medicine technology and primarily relates to a traditional Chinese medicine composition, preparation method, and application for preventing and treating sea cucumber rotten skin syndrome. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 20-80 parts of Terminalia chebula, 3-20 parts of Galla chinensis, 10-50 parts of Andrographis paniculata, and 5-30 parts of Ligusticum chuanxiong. The traditional Chinese medicine composition can significantly enhance the immune function and disease resistance of sea cucumbers, reduce the inflammatory response and pathological damage caused by Vibrio splendens, slow the progression of sea cucumber disease caused by pathogenic bacteria, and reduce the morbidity and mortality of sea cucumbers suffering from rotten skin syndrome, thereby effectively preventing and treating sea cucumber rotten skin syndrome.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition for preventing and treating sea cucumber rot syndrome, a preparation method and application thereof. Background Art

[0002] Sea cucumbers (Apostichopus japonicus), also known as imitation sea cucumbers, possess high nutritional and medicinal value and are a staple species in my country's sea cucumber aquaculture industry. In 2022, my country's farmed sea cucumber production reached approximately 250,000 tons (according to the 2023 China Fisheries Statistical Yearbook). In recent years, with the gradual expansion and increasing intensification of aquaculture operations, coupled with changes in climate and aquaculture water quality, disease problems have become increasingly prominent. Among them, outbreaks of skin ulcer syndrome (SUS) in sea cucumbers cause billions of yuan in economic losses to the industry annually. SUS in sea cucumbers initially manifests as a loss of adhesion, with transparent, loose mouth muscles that fail to close (commonly known as a "swollen mouth"). This is followed by decreased vitality, decreased food intake, increased mucus production, the development of small ulcers, and the discharge of internal organs (commonly known as "vomiting intestines"). Finally, extensive ulceration (commonly known as "skin disintegration") and death occur. This disease is the most common and devastating disease in sea cucumber aquaculture and has become a major bottleneck hindering the stable development of the sea cucumber aquaculture industry. Bacteria are important pathogens causing sea cucumber SUS (Surinary Unsaturated Skin Syndrome), including Vibrio splendidus, Pseudoalteromoonas nigrifaciens, Vibrio alginolyticus, Vibrio harveyi, and Aeromonas salmonicida. SUS is characterized by rapid onset, high infectiousness, and high mortality, resulting in significant economic losses for the sea cucumber aquaculture industry. Currently, there are no nationally approved medications specifically for the prevention and treatment of sea cucumber SUS. The disease is generally treated with a combination of broad-spectrum antibiotics, probiotics, and disinfectants, but the effectiveness is unsatisfactory. Furthermore, the irrational use of chemical drugs can lead to environmental pollution, drug residues, and increased bacterial resistance. Therefore, the development of green and efficient alternative antibiotics has become a crucial task in the prevention and control of sea cucumber diseases.

[0003] Traditional Chinese medicine (TCM) boasts multiple components, multiple targets, and multiple functions. It has been widely used to promote the growth of aquatic animals, enhance immune function, and prevent and treat diseases. TCM must be applied within the guidance of traditional Chinese medicine theory to fully realize its unique strengths. Factors affecting TCM efficacy include the appropriateness of the formula for the underlying condition, the quality of the medicinal ingredients, and the rationality of the preparation process. As the saying goes, "If the preparation is unclear, the medicinal properties will be uncertain, and if the decoction is inaccurate, the condition will not be treated." Among these factors, the quality of the medicinal ingredients can be controlled through quality testing. Prescriptions (or formulas) are the primary means of treating diseases in TCM. Ideal efficacy can be achieved through the principle of monarch, minister, assistant, and guiding ingredients, based on syndrome differentiation and cause determination. Appropriate combinations of medicinal ingredients must be selected, and appropriate dosages, formulations, and methods of administration must be determined based on the principle of combining monarch, minister, assistant, and guiding ingredients. Consequently, "a medicine has its own unique strengths, while a prescription has its own unique efficacy." The preparation process is also one of the most important factors influencing TCM efficacy. The scientific and rational pretreatment of the medicinal ingredients can directly impact the extraction efficiency of the active ingredients and the efficacy of the product. Aquatic animals live in aquatic environments all year round, and their treatment principles, preparation processes and usage techniques of the drugs used are also different from those of terrestrial animals. Therefore, only by organically combining factors such as medicinal material quality, reasonable formulation, medicinal material preparation process and drug usage process can the efficacy of traditional Chinese medicine in the prevention and control of aquatic animal diseases be maximized.

[0004] In the previous research, Wang Yingeng and others from our team developed the "Dual-effect Antibacterial and Immune Compound Chinese Herbal Medicine for Sea Cucumber Skin Putrescence Syndrome" (ZL200910017176.7), which contains Andrographis paniculata, Isatis indigotica, Honeysuckle and Chuanxiong (2:1:3:2). Oral administration has a good effect in preventing and treating Sea Cucumber Skin Putrescence Syndrome. Subsequently, the "A Traditional Chinese Medicine Preparation and Preparation Method for Treating Sea Cucumber Skin Putrescence Syndrome" (ZL 201510257162.8), wherein the Chinese medicine preparation comprises: 25-35 parts of Andrographis paniculata, 15-25 parts of Honeysuckle, 15-25 parts of Scutellaria baicalensis, 25-35 parts of Gallnut, 3 parts of Herba Ophiopogonis, 5 parts of Taraxaci, 4 parts of Herba Psoraleae, 3 parts of Herba Patriniae, 2 parts of Smilax glabra, 5 parts of Flos Chrysanthemi Indici, 2 parts of Radix Sophorae Tonkinensis, 3 parts of Sophorae Flavescentis, 4 parts of Fructus Schizonepetae, 3 parts of Semen Plantaginis, 4 parts of Cortex Ceratitis, 4 parts of Juncus effusus, 2 parts of Bletilla striata, 3 parts of Lotus Root Nodes, and 2 parts of Hedgehog Skin. The preparation process comprises the steps of pulverizing the powder into 300 mesh powder, and the preparation method has a good therapeutic effect on Sea Cucumber Skin Putrescence Syndrome. However, with the emergence of many problems such as global climate change, germplasm degradation, and changes in pathogenic flora characteristics, especially the continuous enhancement of bacterial virulence and the weakening of sea cucumber disease resistance, the pathogenesis of sea cucumber rot syndrome has also changed greatly. In addition, the continuous improvement of the intensive breeding level of sea cucumbers and the diversification of breeding models have placed higher requirements on drugs and their application processes. As a result, it is necessary to redesign, screen and optimize the prevention and control drugs in order to achieve the goals of "preventive treatment" and "curing the disease with medicine". Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a traditional Chinese medicine composition for preventing and treating sea cucumber rot syndrome, a preparation method and application.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a traditional Chinese medicine composition for preventing and treating sea cucumber rot syndrome. The composition comprises the following raw materials in parts by weight: 20-80 parts of Terminalia chebula, 3-20 parts of Galla chinensis, 10-50 parts of Andrographis paniculata, and 5-30 parts of Ligusticum chuanxiong.

[0008] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 30-70 parts of Terminalia chebula, 5-10 parts of Galla chinensis, 15-40 parts of Andrographis paniculata, and 7-20 parts of Ligusticum chuanxiong.

[0009] Preferably, the traditional Chinese medicine composition further comprises the following raw materials in parts by weight: 1-30 parts of Echinacea, 1-30 parts of Sarcandra scabra, and 5-60 parts of Codonopsis pilosula.

[0010] Preferably, the traditional Chinese medicine composition further comprises the following raw materials in parts by weight: 10-25 parts of Echinacea purpurea, 10-25 parts of Sarcandra scabra, and 10-30 parts of Codonopsis pilosula.

[0011] The invention provides a preparation method of the traditional Chinese medicine composition, comprising the following steps: crushing and screening the raw materials, soaking them in water, heating them to a slight boiling point for extraction, filtering and concentrating them to obtain the composition.

[0012] Preferably, before the crushing and screening, the method further comprises any of the following steps: frying the Chuanxiong with rice wine, blanching the Terminalia chebula with sand, and steaming the Echinacea.

[0013] Preferably, the material-liquid ratio of the raw material to water is 1g:5mL to 1g:100mL, and the water is tap water.

[0014] Preferably, the soaking time is 0.5h to 5h, and the extraction time is 1h to 5h.

[0015] Preferably, the frying time of the Chuanxiong is 10 to 30 minutes, the blanching time of the Terminalia chebula is 3 to 10 minutes, and the steaming time of the Echinacea is 10 to 40 minutes.

[0016] The present invention also provides the use of the above-mentioned traditional Chinese medicine composition or the preparation method thereof in preparing a medicine for preventing and treating sea cucumber rotten skin syndrome.

[0017] Beneficial effects of the present invention:

[0018] This prescription is composed of Terminalia chebula, Gallnut, Andrographis paniculata, and Ligusticum chuanxiong, and can be supplemented with Echinacea purpurea, Sarcandra sutchuenensis, and Codonopsis pilosula. Terminalia chebula is astringent and firming as the main herb, while Gallnut is damp-absorbing and sore-healing. Echinacea clears heat and detoxifies, replenishes Qi and strengthens the body's health. Andrographis paniculata and Sarcandra sutchuenensis clear heat and detoxify, cool the blood and eliminate freckles as the auxiliary herb, while Ligusticum chuanxiong is an adjuvant herb that promotes blood circulation and qi. Together, these herbs achieve the effects of astringency, firming, replenishing Qi and strengthening the body's health, clearing heat and detoxifying, cooling the blood and eliminating freckles. This achieves the functions of monarch, minister, adjuvant, and adjuvant, balancing the functions of expelling pathogens and strengthening the body's health. This prescription can significantly enhance the immune function and disease resistance of sea cucumbers, alleviate the inflammatory response triggered by Vibrio splendens, delay the progression of disease caused by pathogenic bacteria, and reduce the morbidity and mortality of sea cucumbers with putrescent skin syndrome, effectively preventing and treating sea cucumber putrescent skin syndrome.

[0019] Based on previous research, the present invention re-screens a traditional Chinese medicine formula with good antibacterial effect through in vitro antibacterial tests, and then prepares it into a preparation according to a scientific production process. The reasonable formula is organically combined with the medicinal material preparation process to achieve the purpose of effectively preventing and treating sea cucumber SUS, providing good technical support for the effective prevention and control of sea cucumber rot skin syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the lysozyme activity of sea cucumber in each experimental group;

[0021] Figure 2 is the superoxide dismutase activity of sea cucumbers in each experimental group;

[0022] Figure 3 is the acid phosphatase activity of sea cucumbers in each experimental group;

[0023] Figure 4 is the alkaline phosphatase activity of sea cucumbers in each experimental group;

[0024] Figure 5 is the incidence of SUS in each group of sea cucumbers after challenge;

[0025] Figure 1-Figure 5 In the table, different lowercase letters indicate significant differences (P<0.05), and different uppercase letters indicate extremely significant differences (P<0.01);

[0026] Figure 6 is the survival rate of each group of sea cucumbers after poison challenge;

[0027] Figure 7 The body wall tissue sections of each group of sea cucumbers, among which 1 is the NC group, 2 is the diseased PC group, 3 is the En group, and 4 is the compound drug group (30 g / kg). The black arrows indicate inflammatory cell infiltration, nuclear condensation and fragmentation, and cell disintegration and necrosis.

[0028] Figure 8The longitudinal muscle tissue sections of each group of sea cucumbers, among which 1 is the NC group, 2 is the diseased PC group, 3 is the En group, and 4 is the compound drug group (30 g / kg). The black arrows indicate the pyknosis of the myofiber nuclei;

[0029] Figure 9 The intestinal tissue sections of each group of sea cucumbers, among which 1 is the NC group, 2 is the diseased PC group, 3 is the En group, and 4 is the compound drug group (30 g / kg). The black arrows indicate necrosis of intestinal mucosal epithelial tissue, with sloughed and necrotic mucosal epithelial cells appearing in the intestinal lumen, forming large gaps.

[0030] Figure 10 The morbidity and mortality rates of sea cucumbers in each group are shown. DETAILED DESCRIPTION

[0031] The invention provides a traditional Chinese medicine composition for preventing and treating sea cucumber rotten skin syndrome. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 20-80 parts of terminalia chebula, 3-20 parts of gallnut, 10-50 parts of andrographis paniculata, and 5-30 parts of ligusticum chuanxiong; preferably, the composition comprises 30-70 parts of terminalia chebula, 5-10 parts of gallnut, 15-40 parts of andrographis paniculata, and 7-20 parts of ligusticum chuanxiong; and more preferably, the composition comprises 45 parts of terminalia chebula, 6 parts of gallnut, 30 parts of andrographis paniculata, and 15 parts of ligusticum chuanxiong.

[0032] Preferably, the traditional Chinese medicine composition further comprises the following raw materials in parts by weight: 1-30 parts of Echinacea, 1-30 parts of Sarcandra, and 5-60 parts of Codonopsis pilosula; more preferably: 10-25 parts of Echinacea, 10-25 parts of Sarcandra, and 10-30 parts of Codonopsis pilosula, further comprising 20 parts of Echinacea, 20 parts of Sarcandra, and 15 parts of Codonopsis pilosula.

[0033] In the present invention, Terminalia chebula: bitter, sour, astringent, flat, enters the large intestine meridian, astringes and strengthens the body, and is mainly used for chronic diarrhea and dysentery, blood in stool and prolapse of the anus. Gallnut: sour, astringent, cold, enters the large intestine meridian, absorbs moisture and heals sores, and is mainly used for carbuncle, sore and ulcer, and wet skin. Andrographis paniculata: bitter, cold, enters the large intestine meridian, clears heat and detoxifies, cools blood, and reduces swelling, and is mainly used for carbuncle, sore and ulcer. Chuanxiong: pungent, warm, enters the liver meridian, promotes blood circulation and qi, dispels wind and relieves pain, and is mainly used for qi and blood stasis, rheumatic pain. Echinacea: clears heat and detoxifies, tonifies qi and strengthens the body. Sarcandra chinensis: bitter, pungent, flat, enters the liver meridian, clears heat and cools blood, promotes blood circulation and eliminates spots, dispels wind and dredges the meridians, and is used for blood-heat purpura. Codonopsis pilosula: sweet, flat, enters the spleen meridian, tonifies the middle and replenishes qi, strengthens the spleen and stomach, and treats poor appetite and diarrhea, fatigue and weakness, and qi deficiency and collapsing. The present invention has no special restrictions on the sources of the Terminalia chebula, Gallnut, Andrographis paniculata, Ligusticum chuanxiong, Echinacea purpurea, Sarcandra scabra and Codonopsis pilosula, and conventional commercially available medicinal pieces that meet national standards can be used.

[0034] The present invention also provides a preparation method of the above-mentioned traditional Chinese medicine composition, comprising the following steps: crushing and sieving the raw materials, soaking them in water, heating them to a slight boiling point for extraction, filtering and concentrating them to obtain the composition.

[0035] Preferably, before crushing and screening, the process further comprises any of the following steps: the Chuanxiong is stir-fried with yellow rice wine to enhance its effects of promoting blood circulation and qi, and dispersing wind; the Terminalia chebula is blanched with sand to enhance its astringent and firming effect; the Echinacea is steamed to effectively improve its effects of clearing heat and detoxifying, and replenishing qi and strengthening the body. The stir-frying time of the Chuanxiong is preferably 10 to 30 minutes, more preferably 15 to 25 minutes, and further 20 minutes; the blanching temperature of the Terminalia chebula is preferably 100-350°C, more preferably 200 to 300°C, and further 250°C; the blanching time of the Terminalia chebula is preferably 3 to 10 minutes, more preferably 4 to 8 minutes, and further 5 minutes; the Echinacea is preferably steamed by adding the medicinal materials when the water is near boiling, and the steaming time is preferably 10 to 40 minutes, more preferably 15 to 30 minutes, and further 20 minutes.

[0036] In the present invention, the raw materials are pulverized and then screened. The present invention does not specifically limit the pulverization method; conventional pulverization methods in the art can be used. The sieving is preferably performed through a 100-200 mesh sieve, more preferably through a 120-180 mesh sieve, and further preferably through a 150 mesh sieve. The pulverization of the raw materials accelerates the release of the active ingredients and improves the extraction efficiency of the active ingredients, thereby reducing the amount of raw materials used, shortening the extraction time, and saving energy.

[0037] In the present invention, the sieved raw materials are soaked in water. The material-liquid ratio of the raw materials to water is preferably 1g:5mL to 1g:100mL, more preferably 1g:10mL to 1g:80mL, and further 1g:70mL. The water is preferably tap water. The soaking time is preferably 0.5h to 5h, more preferably 2h to 4h, and further 3h. In the present invention, after the raw materials are soaked, the water in which the raw materials are soaked is heated to a slight boiling point for extraction. The extraction time is preferably 1h to 5h, more preferably 1.5 to 3h, and further 2h.

[0038] In the present invention, the raw materials are extracted and then filtered and concentrated to obtain the Chinese medicine composition of the present invention. The present invention has no particular limitation on the methods of filtration and concentration, and conventional methods in the art can be used.

[0039] The present invention also provides the use of the aforementioned traditional Chinese medicine composition or its preparation method in the preparation of a medicament for the prevention and treatment of sea cucumber rotten skin syndrome. Symptoms such as loss of adhesion, loose oral muscles and inability to close, head shaking, and internal discharge in sea cucumbers infected with Vibrio splendens indicate a weak exterior and Qi stagnation. Extensive ulceration on the sea cucumber surface suggests accumulation of dampness and heat. When the Chinese medicine composition of the present invention is used for treatment, the terminalia chebula and the gallnut are used for astringency and firming, so as to astringe dissipation, consolidate slippage and reduce vital energy loss, absorb moisture and consolidate ulcers to eliminate moisture, astringe wounds and alleviate infection, the echinacea is used for clearing away heat and detoxifying, replenishing qi and strengthening the vital energy, the andrographis paniculata and the scutellaria baicalensis are used for cooling blood and eliminating freckles, and the ligusticum chuanxiong is used for promoting blood circulation and promoting qi, thereby achieving the effects of astringency and firming, replenishing qi and strengthening the vital energy, clearing heat and detoxifying, cooling blood and eliminating freckles, achieving both the functions of expelling pathogens and strengthening the vital energy, and thus significantly improving the immune function and disease resistance of the sea cucumber, reducing the body's inflammatory response and tissue pathological damage caused by Vibrio splendens, delaying the progression of the disease of the sea cucumber caused by pathogenic bacteria, reducing the morbidity and mortality of the sea cucumber rotten skin syndrome, and having the effect of effectively preventing and treating the sea cucumber rotten skin syndrome.

[0040] In the present invention, the drug for preventing and treating sea cucumber rotten skin syndrome comprises the pharmaceutical composition of the present invention as an active ingredient and also comprises pharmaceutically acceptable excipients.

[0041] The present invention has no particular limitation on the dosage form of the drug, and conventional dosage forms in the art can be used. The present invention has no particular limitation on the route of administration of the drug, and preferably the drug is directly soaked in the sea cucumber or mixed with feed and fed to the sea cucumber.

[0042] In the present invention, the method of using the above-mentioned drug is: when used for preventing or treating sea cucumber rot syndrome, the above-mentioned drug is mixed with bait and fed every day, with a dosage of 30-40g / kg feed, and is used for 10 consecutive days.

[0043] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] In the following examples, unless otherwise specified, all methods are conventional.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0046] In a specific embodiment of the present invention:

[0047] Medicinal materials: Terminalia chebula, Gallnut, Andrographis paniculata, Ligusticum chuanxiong, Echinacea purpurea, Sarcandra scabra, Codonopsis pilosula and other Chinese herbal medicine slices were purchased from Hebei Quantai Pharmaceutical Co., Ltd.

[0048] Control drug: Enrofloxacin powder for aquatic products, purchased from a GMP veterinary drug factory in Shandong, with a content specification of 5%. Preliminary drug sensitivity tests showed that Vibrio splendens was extremely sensitive to enrofloxacin (the diameter of the inhibition zone reached 28.07±0.76mm), so it was selected as the control drug for the Chinese medicine composition.

[0049] Experimental strain: Vibrio splendens KL-1 strain was isolated from a typical SUS-infected sea cucumber by the Disease Research Laboratory of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. A single purified strain was selected and inoculated into TSB liquid medium. The culture was incubated in a shaking incubator at 28°C and 180 rpm for 12 to 16 hours. The bacterial culture in the logarithmic growth phase was collected and the OD 600 The value is 0.5~0.7 (the bacterial concentration is about 10 7 CFU / mL) were used for the test.

[0050] Experimental Animals: Sea cucumbers were purchased from the farm of Qingdao Ruizi Group Development Co., Ltd. Healthy individuals of uniform size (55 ± 10 g / head) were selected and temporarily housed in 300 L aquaria for 5 days before use in this experiment. The experimental water used was clean natural seawater, maintained at a temperature of 16.5°C to 18.2°C, a pH of 7.0 to 8.5, a salinity of 25 to 32, and a dissolved oxygen level of ≥5.0 mg / L. During the acclimation period, they were fed a light diet, with water changes of approximately one-third daily. Full experiments were conducted after 5 days of acclimation.

[0051] Reagents and consumables: The kits used for immunoenzyme activity assays of lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP), and alkaline phosphatase (AKP) were purchased from Nanjing Jiancheng Bioengineering Institute.

[0052] Data processing: SPSS 26.0 software was used to perform statistical analysis on the experimental data. The data were first subjected to one-way ANOVA, and the results were expressed as "mean ± standard deviation (mean ± SD)".

[0053] Example 1

[0054] Terminalia chebula, Galla chinensis, Andrographis paniculata, and Ligusticum chuanxiong were used as the four factors in the orthogonal experiment. The dosage of each raw material was set at three levels (Table 1). 4 ) were constructed to obtain 9 formulas (FF-1 to FF-9).

[0055] Table 1 Level table of compound compatibility factors in orthogonal design (g)

[0056]

[0057] The raw materials of compound FF-1 to FF-9 were crushed and sieved through 150 mesh respectively, and mixed according to the proportion. 5 g of medicinal material powder was weighed and added to room temperature tap water at a ratio of 1 g:70 mL. The mixture was soaked for 3 h, heated to a slight boiling point and extracted for 2 h. The mixture was filtered and the filtrate was concentrated under reduced pressure to a medicinal solution with a crude drug content of 500 mg / mL. The mixture was sterilized at 121°C for 20 min, packaged after cooling, and stored at 4°C for later use.

[0058] Example 2

[0059] Prescription: Terminalia chebula 45g, Gallnut nut 6g, Andrographis paniculata 30g and Ligusticum chuanxiong 15g.

[0060] The Chinese medicine was crushed with a grinder and sieved with 150 mesh. 5 g of the medicinal material powder was weighed and added to room temperature tap water at a ratio of 1 g:70 mL. The mixture was soaked for 3 h, heated to a slight boiling point and extracted for 2 h. The mixture was filtered and the filtrate was concentrated under reduced pressure to a medicinal solution with a crude drug content of 500 mg / mL. The mixture was sterilized at 121 °C for 20 min, cooled, and packaged. The mixture was stored at 4 °C for later use.

[0061] Example 3

[0062] Prescription: Terminalia chebula 80g, Gallnut 20g, Andrographis paniculata 50g and Ligusticum chuanxiong 30g.

[0063] The Chinese medicine was crushed with a grinder and sieved with 100 mesh. 5 g of the medicinal material powder was weighed and added to room temperature tap water at a ratio of 1 g:5 mL. The mixture was soaked for 0.5 h, heated to a slight boiling point for extraction for 1 h, filtered, and the filtrate was concentrated under reduced pressure to a medicinal solution with a crude drug content of 1500 mg / mL. The solution was sterilized at 121°C for 20 min, cooled, and packaged. The solution was stored at 4°C for later use.

[0064] Example 4

[0065] Prescription: Terminalia chebula 20g, Gallnut nut 3g, Andrographis paniculata 10g and Ligusticum chuanxiong 5g.

[0066] The Chinese medicine was crushed with a grinder and sieved with 200 mesh. 5 g of the medicinal material powder was weighed and added to room temperature tap water at a ratio of 1 g:100 mL. The mixture was soaked for 5 h, heated to a slight boiling point and extracted for 5 h. The mixture was filtered and the filtrate was concentrated under reduced pressure to a medicinal solution with a crude drug content of 250 mg / mL. The mixture was sterilized at 121°C for 20 min, packaged after cooling, and stored at 4°C for later use.

[0067] Example 5

[0068] The only difference from Example 2 is that the medicinal solution is prepared with a crude drug content of 1 g / mL, and the rest is the same as Example 2.

[0069] Example 6

[0070] The only difference from Example 2 is that the Terminalia chebula and Ligusticum chuanxiong are further processed before being crushed and sieved. Ligusticum chuanxiong is stir-fried with rice wine for 20 minutes, and Terminalia chebula is scalded with sand at 250° C. for 5 minutes. The rest is the same as Example 2.

[0071] Example 7

[0072] The only difference from Example 5 is that the Terminalia chebula and Ligusticum chuanxiong are further processed before crushing and sieving. Ligusticum chuanxiong is stir-fried with rice wine for 20 minutes, and Terminalia chebula is scalded with sand at 250° C. for 5 minutes. The rest are the same as Example 5.

[0073] Example 8

[0074] The only difference from Example 5 is that the Terminalia chebula and Ligusticum chuanxiong are further processed before being crushed and sieved. Ligusticum chuanxiong is stir-fried with rice wine for 10 minutes, and Terminalia chebula is scalded with sand at 350° C. for 3 minutes. The rest is the same as Example 5.

[0075] Example 9

[0076] The only difference from Example 5 is that the Terminalia chebula and Ligusticum chuanxiong are further processed before being crushed and sieved. Ligusticum chuanxiong is stir-fried with rice wine for 30 minutes, and Terminalia chebula is scalded with sand at 100° C. for 10 minutes. The rest are the same as Example 5.

[0077] Example 10

[0078] The only difference from Example 7 is that 20 g of Echinacea, 20 g of Sarcandra scutellariae, and 15 g of Codonopsis pilosula are further added to the prescription, wherein the Echinacea is further processed before being crushed, specifically, the Echinacea is added when the water is near boiling and steamed for 20 minutes. The rest is the same as in Example 7.

[0079] Example 11

[0080] The only difference from Example 3 is that 30 g of Echinacea, 30 g of Sarcandra scutellariae, and 60 g of Codonopsis pilosula are further added to the prescription, wherein the Echinacea is further processed before being crushed, specifically, the Echinacea is added when the water is near boiling and steamed for 10 minutes. The rest is the same as in Example 3.

[0081] Example 12

[0082] The only difference from Example 4 is that 1 g of Echinacea, 1 g of Sarcandra scabra, and 5 g of Codonopsis pilosula are further added to the prescription, wherein Echinacea is further processed before being crushed, specifically, Echinacea is added when the water is near boiling and steamed for 40 minutes. The rest is the same as in Example 4.

[0083] Experimental Example 1

[0084] Screening of dosage ratio of Chinese medicine combination

[0085] The nine formulations (FF-1 to FF-9) in Example 1 were used as experimental drugs to determine the diameter of the inhibition zone, the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC) of each formulation. The orthogonal experiment was repeated three times.

[0086] Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination method: The MIC values ​​of the 9 formulations against Vibrio splendens were determined using the modified microdilution method. 100 μL of TSB medium was added to each well of a 96-well cell culture plate. 100 μL of the drug solution of the formulation to be tested was added to the first row, and the solution was diluted sequentially to a final concentration of 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 mg / mL of drug-containing medium. 100 μL of bacterial solution (10 7 CFU / mL), and finally add 10μL of 1% red tetrazolium solution to each well, with 3 replicates per group. The last 2 columns are control groups: positive control (culture medium + bacteria) and negative control (culture medium + culture medium / sterile water). Culture in a constant temperature incubator at 28°C. When the positive control group shows a color change, record the drug concentration corresponding to the previous row of color-changing wells in each column, which is the MIC. Pipette 100μL of liquid from the above-mentioned uncolored wells, apply them to sterile TSB solid culture medium plates and mark the corresponding drug concentrations. Culture them upside down in a constant temperature incubator at 28°C for 24 hours. The concentration corresponding to the plate where no bacterial growth occurs is the MBC.

[0087] The experimental results are shown in Table 2. The results showed that A3B1C1D1 had the largest inhibition zone diameter, indicating that formulation FF-Z (Terminalia chebula:Gallacus chinensis:Andrographis paniculata:Ligusticum chuanxiong = 15:2:10:5) had the best antibacterial effect. After weighing the medicinal materials according to the above ratio and extracting them (the solution of Example 2), in vitro antibacterial testing was conducted. The results showed that formulation FF-Z (the solution of Example 2) had an inhibition zone diameter of 23.10±0.62 mm, and MIC and MBC values ​​of 7.81 mg / mL and 15.63 mg / mL, respectively, showing the best antibacterial effect against Vibrio splendens among all the tested drugs. Therefore, formulation FF-Z was selected for further in vivo pharmacodynamic studies.

[0088] Table 2 Inhibition zone diameter, MIC and MBC of each formula

[0089]

[0090] Experimental Example 2

[0091] Preventive effect of Chinese herbal medicine combination on sea cucumber rotten skin syndrome

[0092] Experimental groups and treatments:

[0093] Medicated feed: 10, 20, 30, and 40 mL of the medicinal solution prepared in Example 5 were sprayed evenly onto 1 kg of sea cucumber feed, then fully bonded with an alginic adhesive (sodium alginate: water = 10 g: 90 mL) and dried to obtain feeds containing 10 g / kg, 20 g / kg, 30 g / kg, and 40 g / kg of the crude drug, respectively. A total of 420 healthy sea cucumbers of similar size, after being temporarily raised for 5 days, were randomly divided into seven groups: a negative control group (NC group, ordinary feed), a positive control group (PC group, ordinary feed), a compound drug group (10 g / kg, 20 g / kg, 30 g / kg, and 40 g / kg medicated feed groups), and a drug control group (En group, enrofloxacin powder administered at 20 g / kg feed). Each group consisted of three replicates, with 20 sea cucumbers per replicate placed in a 120 L water tank (90 L of seawater) for testing. Each group was fed once a day, with 1% of the sea cucumber body weight each time, for 10 consecutive days.

[0094] On the 0th, 4th, 7th and 10th day of administration, three sea cucumbers were randomly selected from each group for sampling. The body surface was wiped with sterile gauze and placed on ice. 1 mL of the sea cucumber coelomic fluid was extracted into a centrifuge tube and centrifuged at 4°C, 5000 r / min for 10 min. The supernatant was used for the determination of LZM, SOD, ACP and AKP.

[0095] Toxic challenge experiment: After stopping the drug and sampling, the remaining sea cucumbers in each experimental group, except the NC group, were incubated with Vibrio splendens at a final concentration of 10 7 The animals were immersed in 60 L of aquaculture water containing 100 CFU / mL for 24 hours. They were then fed a standard diet daily with regular bottom water changes. The incidence of rotting skin syndrome, symptoms, and mortality were recorded for 20 consecutive days. At the end of the experiment, five sea cucumbers from each group were dissected and the body wall, longitudinal muscle, and intestine were fixed in fixative for histopathological examination.

[0096] Experimental results:

[0097] 1. Effects of Chinese herbal medicine combination on humoral immune enzyme activities in sea cucumbers

[0098] The sea cucumbers were fed with different doses of compound FF-Z. The coelomic fluid of the sea cucumbers was collected on the 0th, 4th, 7th and 10th day after the start of feeding. The effect of compound FF-Z on LZM in the coelomic fluid of the sea cucumbers was tested. The results are shown in Figure 3. Figure 1As shown in the figure. Sea cucumber LZM content was high before the experiment began. Within 10 days of dosing, LZM levels in sea cucumbers in the different dose groups of compound FF-Z showed varying degrees of change, generally showing a downward and then upward trend. At 4 days of dosing, LZM activity was higher in sea cucumbers in the En group and the 20 g / kg compound FF-Z group, but there were no significant differences from other groups (P>0.05). At 10 days of dosing, LZM levels reached their highest values ​​in the 30 g / kg and 40 g / kg compound FF-Z groups, but there were no significant differences from the other groups (P>0.05).

[0099] Compound FF-Z has a significant effect on the SOD activity of sea cucumber ( Figure 2 As the drug feeding time increased, the SOD activity of sea cucumbers in the groups receiving different doses of compound FF-Z gradually increased. The SOD activity of sea cucumbers in the group receiving 30 g / kg compound FF-Z was significantly higher than that in the other groups on the 4th, 7th, and 10th days (P < 0.01). On the 10th day, the SOD activity of sea cucumbers in the groups receiving different doses of compound FF-Z reached its maximum and was significantly higher than that in the NC group.

[0100] The effects of different doses of compound FF-Z on the ACP activity of sea cucumber coelomic fluid varied greatly in different time periods ( Figure 3 The ACP activity of sea cucumbers in the FF-Z 10 g / kg compound group reached its highest value on the 4th day of administration, and was significantly different from that in the NC group (P<0.01). The ACP activity of sea cucumbers in the FF-Z 20 g / kg compound group reached its highest value on the 7th day of administration, and was significantly different from that in the other groups (P<0.01). The ACP activity of sea cucumbers in the FF-Z 30 g / kg compound group and the FF-Z 40 g / kg compound group showed a trend of first decreasing and then increasing with the extension of feeding time, and reached its highest value on the 10th day of administration, at which time the FF-Z 30 g / kg compound group was significantly different from that in the other groups (P<0.01).

[0101] Depend on Figure 4The effects of the compound Chinese medicine on AKP activity in the coelomic fluid of sea cucumbers varied significantly over time. AKP activity in the 10 g / kg, 20 g / kg, and 30 g / kg FF-Z groups increased, then decreased, and then increased again with prolonged feeding time, whereas AKP activity in the 40 g / kg FF-Z group decreased, then increased. On day 4 of administration, AKP activity in the 10 g / kg, 20 g / kg, 30 g / kg, and En groups was significantly higher than that in the NC group (P < 0.01). On day 10 of administration, AKP activity in the four FF-Z dose groups was significantly higher than that in the NC and En groups. AKP activity in the FF-Z dose groups was positively correlated with the dose of the Chinese medicine.

[0102] 2. Experiment on Sea Cucumber Skin Rot Syndrome Caused by Toxin

[0103] After the preventive medication period, all sea cucumbers in the NC group, except the NC group, were challenged with Vibrio splendens by immersion for 24 hours and observed for 20 consecutive days. Five days after challenge, the PC group began to show early symptoms of SUS, manifested by loss of adhesion and a transparent, loose mouth muscle that failed to close. Seven days after challenge, sea cucumbers showed decreased vitality, decreased food intake, increased mucus production, and the development of small ulcers, with an incidence rate of 19.64%. Ten days after challenge, some sea cucumbers developed extensive ulcers and died, with a cumulative incidence of 63.69%. Twenty days after challenge, the cumulative incidence in the PC group reached 84.45%. Throughout the experimental period, the NC group remained normal, with no cases of illness or mortality, confirming the success of the V. splendens immersion infection.

[0104] 3. Effects of Chinese herbal medicine combination on the morbidity and survival rate of sea cucumbers

[0105] After stopping the drug and immersing in the bath to challenge the poison, each group of sea cucumbers were observed for 20 consecutive days. Figure 5 Five days after the challenge, the En group developed SUS symptoms similar to those of the PC group. Seven days after the challenge, sea cucumbers in the four FF-Z compound dose groups began to develop SUS symptoms, but the onset time was later and the symptoms were milder than those in the PC group. The incidence rates of sea cucumbers in the FF-Z compound 20 g / kg group, the FF-Z compound 30 g / kg group, and the FF-Z compound 40 g / kg group were significantly lower than those in the PC and En groups (P < 0.05). Twenty days after the challenge, the incidence rate in the En group was 78.75%, while the incidence rates in the FF-Z compound 20 g / kg group, the FF-Z compound 30 g / kg group, and the FF-Z compound 40 g / kg group were 22.08%, 22.14%, and 14.58%, respectively, which were all significantly lower than those in the PC and En groups (P < 0.05).

[0106] The statistical results of the survival rates of each group showed that the sea cucumbers began to die 10 days after the challenge, and the peak period of death due to rotten skin syndrome was 11-15 days after the challenge. 16 days after the challenge, the survival rate of the sea cucumbers tended to be stable. The survival rates of the various dose groups of compound FF-Z were higher than 78.61% of the PC group and 87.5% of the En group. Among them, the survival rate of the 40g / kg compound FF-Z group was the highest, at 97.92%. The survival rates of the other three dose groups of compound FF-Z were all 93.75%. 20 days after the challenge, the survival rates of the various groups remained unchanged. For details, see Figure 6 .

[0107] 4. Histopathological Observation

[0108] By analyzing the enzyme activity, morbidity, survival rate and other indicators of the sea cucumbers in each experimental group, it was found that the efficacy of the compound FF-Z at doses of 20g / kg, 30g / kg and 40g / kg was relatively good, among which the compound FF-Z at doses of 30g / kg and 40g / kg had better efficacy. Based on production cost considerations, the body wall, longitudinal muscle and intestine of the sea cucumbers in the compound FF-Z 30g / kg group and the NC group, PC group and En group were selected for tissue section observation. The results are shown in Figure 2. Figure 7 、 Figure 8 、 Figure 9 As shown. Observations have shown that after infection with Vibrio splendens, a large number of inflammatory cells aggregate in the body wall of the diseased sea cucumber, forming an inflammatory reaction zone, which leads to nuclear condensation, lysis, and dark staining of some cells ( Figure 7 ); Necrotic lesions appear in the muscles of diseased sea cucumbers, mainly manifested by the fragmentation of the fascicle membrane, the enlargement of the gaps between the fascicles and the appearance of a large number of tissue fragments ( Figure 8 ); The intestinal mucosal epithelium of diseased sea cucumbers showed superficial necrosis, with swollen and disintegrated cell nuclei and necrotic and detached epithelial cells ( Figure 9 The body wall, longitudinal muscles, and intestinal tissues of the NC group were neatly arranged, uniformly colored, and exhibited essentially normal tissue structure. Under drug intervention, the severity of infection and inflammatory responses in sea cucumber tissues were less severe than in the PC group, and the tissue health of the 30g / kg FF-Z compound group was superior to that of the En group. This suggests that the FF-Z compound can, to a certain extent, alleviate tissue inflammation caused by Vibrio splendens infection and is more effective than enrofloxacin powder for aquaculture.

[0109] The above experimental results show that the Chinese medicine composition of the present invention has a good preventive effect on sea cucumber skin rot syndrome caused by Vibrio splendens, and can improve the immune function and disease resistance of sea cucumbers, reduce the inflammatory response caused by Vibrio splendens, and delay the progression of sea cucumber disease caused by pathogenic bacteria.

[0110] Experimental Example 3

[0111] Comparison of the efficacy of raw materials prepared by different methods

[0112] In vitro antibacterial experiments were conducted using the two traditional Chinese medicine compound solutions of Example 2 and Example 6. Referring to the Oxford cup method, a 90 mm diameter culture plate was placed in a clean bench and 7 to 9 ml of TSB culture medium (1.5% agar) melted by heating was poured into it. After evenly spreading and letting it stand to solidify, the Oxford cup was placed in equal parts on the plate, and then 10 to 15 ml of TSB culture medium (1% agar) melted by heating was poured into it. After ultraviolet sterilization, the Oxford cup was removed with sterile tweezers to form a culture medium plate with holes. 100 μL (1×10 7 CFU / mL), and evenly spread it on the well plate with a sterile cotton swab, then add 100 μL of the Chinese medicine compound solution to the well, place it upright in a 28°C constant temperature incubator for overnight culture for 16 hours, and measure the diameter of the inhibition zone with a vernier caliper three times. The average value was used to determine the antibacterial ability of the drug. The judgment standard was: the inhibition zone diameter ≥ 20 mm was extremely sensitive "+++", 15 mm ≤ inhibition zone diameter < 20 mm was highly sensitive "++", 10 mm ≤ inhibition zone diameter < 15 mm was moderately sensitive "+", and the inhibition zone diameter < 10 mm was low sensitive or ineffective "-".

[0113] Table 3 Comparison of in vitro antibacterial test results of different Chinese herbal compound liquids

[0114]

[0115] The experimental results showed that the medicinal solution of Example 6 had a better effect in inhibiting Vibrio splendens, indicating that frying Chuanxiong with rice wine for 20 minutes and scalding Terminalia chebula with sand for 5 minutes before crushing and extracting had a positive effect on improving the efficacy.

[0116] Experimental Example 4

[0117] Therapeutic effect of traditional Chinese medicine combination on sea cucumber rotten skin syndrome

[0118] Medicated feed was prepared using the drug solutions of Example 7 (Formulation 1) and Example 10 (Formulation 2) as test drugs: 30 mL of each test drug solution was evenly sprayed on 1 kg of sea cucumber feed, and then fully bonded with an alginic adhesive (sodium alginate: water = 10 g: 90 mL) and dried to obtain 30 g / kg of medicated feed for later use.

[0119] 120 healthy sea cucumbers were randomly divided into 4 groups, namely prescription group 1, prescription group 2, positive control group and blank control group, with 30 sea cucumbers in each group. 7 CFU / mL of seawater was immersed in the poison for 24 hours. At the same time, the animals were fed with the mixture of prescription 1 and prescription 2 for 10 consecutive days. After stopping the drug, the animals were observed for another 10 days.

[0120] The experimental results are shown in Figure 10By the end of the observation period, the morbidity rate in the positive control group was 86.7%, and the mortality rate reached 16.7%. The sea cucumbers in the blank control group were normal, with no illness or mortality, demonstrating the successful replication of this challenge infection model. The morbidity rate in group 1 was 33.3%, with a mortality rate of 10.0%. The morbidity rate in group 2 was 26.7%, with a mortality rate of 6.7%. The experimental results show that both prescriptions 1 and 2 have good therapeutic effects on sea cucumber rot skin syndrome. At the same time, the experimenters found that in this experimental case, the feeding and vitality of the sea cucumbers in prescription 2 were better than those in prescription 1, proving that prescription 2 has a more ideal therapeutic effect on sea cucumber rot skin syndrome. That is, before preparing the extract of the Chinese medicine composition, the Chuanxiong was stir-fried with rice wine for 20 minutes, the terminalia chebula was scalded for 5 minutes, and the Echinacea was steamed for 20 minutes when the water was near boiling, and then crushed and extracted. More active ingredients can be obtained, and the therapeutic effect on sea cucumber rot skin syndrome is better.

[0121] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for preventing and treating sea cucumber rot syndrome, characterized in that: The invention is prepared from the following raw materials in parts by weight: 20-80 parts of Terminalia chebula, 3-20 parts of Galla chinensis, 10-50 parts of Andrographis paniculata and 5-30 parts of Ligusticum chuanxiong.

2. The Chinese medicine composition according to claim 1, characterized in that The invention is prepared from the following raw materials in parts by weight: 30-70 parts of Terminalia chebula, 5-10 parts of Galla chinensis, 15-40 parts of Andrographis paniculata and 7-20 parts of Ligusticum chuanxiong.

3. A Chinese medicine composition for preventing and treating sea cucumber rot syndrome, characterized in that: The invention is prepared from the following raw materials in parts by weight: 20-80 parts of Terminalia chebula, 3-20 parts of Galla chinensis, 10-50 parts of Andrographis paniculata, 5-30 parts of Chuanxiong rhizome, 1-30 parts of Echinacea purpurea, 1-30 parts of Sarcandra scabra and 5-60 parts of Codonopsis pilosula.

4. The Chinese medicine composition according to claim 3, characterized in that The invention is prepared from the following raw materials in parts by weight: 30-70 parts of Terminalia chebula, 5-10 parts of Gallnut, 15-40 parts of Andrographis paniculata, 7-20 parts of Chuanxiong, 10-25 parts of Echinacea purpurea, 10-25 parts of Sarcandra scabra and 10-30 parts of Codonopsis pilosula.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The following steps are involved: The raw materials are crushed and sieved, soaked in water, heated to slightly boiling point for extraction, filtered and concentrated to obtain the product.

6. The preparation method according to claim 5, characterized in that Before crushing and screening, the method further comprises any of the following steps: frying the Chuanxiong with rice wine; blanching the Terminalia chebula with sand; and steaming the Echinacea.

7. The preparation method according to claim 5 or 6, characterized in that: The material-liquid ratio of the raw material to water is 1g:5mL~1g:100mL, and the water is tap water.

8. The preparation method according to claim 5 or 6, characterized in that: The soaking time is 0.5h~5h, and the extraction time is 1h~5h.

9. The preparation method according to claim 6, characterized in that The stir-frying time of the Chuanxiong is 10-30 minutes, the blanching time of the Terminalia chebula is 3-10 minutes, and the steaming time of the Echinacea is 10-40 minutes.

10. Use of the traditional Chinese medicine composition according to any one of claims 1 to 4 or the preparation method according to any one of claims 5 to 9 in preparing a medicament for preventing and treating sea cucumber rot syndrome.

Images