Fermented traditional Chinese medicine preparation for protecting liver and promoting bile secretion of aquatic animals and preparation method thereof

Abstract

The application provides a fermented traditional Chinese medicine preparation for protecting liver and promoting bile secretion of aquatic animals and a preparation method, and relates to the field of disease prevention and treatment of aquatic animals.The fermented traditional Chinese medicine preparation is made from ten kinds of traditional Chinese medicines, i.e.gentian, wormwood, sedum lineare, lobelia, gambir plant, dendrobium, radix paeoniae alba, gynostemma pentaphyllum, medlar and polygonatum, and has a synergistic effect through scientific compounding.Meanwhile, alkaline protease is added to promote digestion and absorption, and brewer's yeast and bacillus licheniformis are added to improve the intestinal microecological environment.The in-vitro test result shows that the fermented traditional Chinese medicine preparation can effectively inhibit the oxidative stress reaction induced by TGF-beta and has a liver protection effect.The in-vivo test result shows that the fermented traditional Chinese medicine preparation can effectively relieve liver lipid peroxidation, promote bile secretion and reduce bile stasis, and provides a fermented traditional Chinese medicine preparation with double effects of protecting liver and promoting bile secretion for the aquaculture industry.

Description

Technical Field

[0001] This invention relates to the field of disease prevention and control in aquatic animals, specifically to a fermented traditional Chinese medicine preparation for protecting the liver and promoting bile secretion in aquatic animals and its preparation method. Background Technology

[0002] With the development of modern agriculture and the advancement of technology, aquaculture has achieved rapid growth. However, high-density intensive farming has led to the deterioration of the aquatic environment and frequent outbreaks of aquatic diseases. Hepatobiliary diseases have become a key issue restricting the profitability of aquaculture, mainly manifested as hepatic steatosis, gallbladder enlargement, and metabolic dysfunction, severely impairing the healthy growth of aquatic animals. As vital metabolic and hematopoietic organs, the condition of the liver and gallbladder directly affects the body's immunity and growth performance. Currently, the prevention and treatment of hepatobiliary diseases in aquatic animals mainly rely on chemical drugs. Long-term use can lead to increased drug resistance, drug residues polluting water bodies, reducing the nutritional value of aquatic products, and even endangering human health.

[0003] Traditional Chinese medicine (TCM) has shown promising application prospects in the prevention and treatment of diseases in aquatic animals due to its natural, low-toxicity, and residue-free characteristics. While traditional hepatoprotective and choleretic TCMs (such as Artemisia capillaris, Bupleurum chinense, and Scutellaria baicalensis) possess the effects of clearing heat and detoxifying, and soothing the liver and promoting bile secretion, single-herb TCM components suffer from limited target sites and low bioavailability. Existing compound preparations enhance efficacy by combining multiple TCM components, but still face challenges such as insufficient release of active ingredients and single-target regulation. Therefore, developing a fermented TCM preparation that integrates efficient release of active ingredients and multi-target regulation is of great significance for promoting green aquaculture. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a fermented traditional Chinese medicine preparation for liver protection and choleretic purposes in aquatic animals, along with its preparation method.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a fermented traditional Chinese medicine preparation for protecting the liver and promoting bile secretion in aquatic animals. The fermented traditional Chinese medicine preparation is made from the following traditional Chinese medicine raw materials in parts by weight: 15-35 parts of Gentiana scabra, 12-32 parts of Artemisia capillaris, 10-30 parts of Sedum sarmentosum, 8-28 parts of Lobelia chinensis, 5-25 parts of Angelica sinensis, 3-22 parts of Dendrobium nobile, 3-20 parts of Paeonia lactiflora, 3-20 parts of Gynostemma pentaphyllum, 3-20 parts of Lycium barbarum, and 4-22 parts of Polygonatum sibiricum.

[0009] Preferably, the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 20-30 parts of gentian root, 17-27 parts of artemisia capillaris, 15-25 parts of sedum sarmentosum, 13-23 parts of lobelia chinensis, 10-20 parts of angelica sinensis, 7-17 parts of dendrobium, 5-15 parts of white peony root, 3-13 parts of gynostemma pentaphyllum, 5-15 parts of wolfberry, and 7-17 parts of polygonatum sibiricum.

[0010] Preferably, the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 25 parts of gentian root, 22 parts of artemisia capillaris, 20 parts of sedum sarmentosum, 18 parts of lobelia chinensis, 15 parts of angelica sinensis, 12 parts of dendrobium, 10 parts of white peony root, 8 parts of gynostemma pentaphyllum, 10 parts of wolfberry, and 12 parts of polygonatum sibiricum.

[0011] Furthermore, the fermented traditional Chinese medicine preparation also includes alkaline protease at a weight ratio of 15%-25% of the raw materials, brewer's yeast liquid at a weight ratio of 15%-30%, and Bacillus licheniformis liquid at a weight ratio of 3%-15%.

[0012] Furthermore, the fermented traditional Chinese medicine preparation also includes 20% alkaline protease, 22% brewer's yeast liquid, and 10% Bacillus licheniformis liquid, accounting for 20% of the weight of the traditional Chinese medicine raw materials.

[0013] This invention provides a method for preparing the fermented traditional Chinese medicine preparation, comprising the following steps:

[0014] (1) Weigh each Chinese herbal raw material according to the above weight proportions, wash and dry it, then pulverize it through a 100-mesh sieve. Mix the obtained powder with 4-8 times the weight of deionized water and stir to form a homogeneous suspension.

[0015] (2) Add alkaline protease to the suspension, adjust the pH to 6.5-7.5, and carry out the enzymatic hydrolysis reaction at 40-45℃ for 4-6 h. After the reaction is completed, filter and collect the hydrolysate.

[0016] (3) Activation of the strains: Saccharomyces cerevisiae was cultured in malt extract liquid medium at 30°C with shaking for 24 h, and Bacillus licheniformis was cultured in LB liquid medium at 37°C with shaking for 12 h to obtain Saccharomyces cerevisiae and Bacillus licheniformis liquid cultures, with a strain concentration of ≥1×10⁻⁶. 8 CFU / mL;

[0017] (4) Add 2wt% glucose and 1.5wt% peptone to the enzymatic hydrolysate as fermentation substrate, inoculate with Saccharomyces cerevisiae liquid and Bacillus licheniformis liquid according to the above weight ratio, control the fermentation temperature at 30-35℃, maintain the pH value at 5.5-6.5, carry out anaerobic / microaerobic fermentation for 5-7 days, filter the fermentation liquid, concentrate under reduced pressure, and then spray dry and granulate to obtain the fermented Chinese medicine preparation of the present invention.

[0018] This invention provides the application of the fermented traditional Chinese medicine preparation in the preparation of hepatoprotective and choleretic drugs for aquatic animals.

[0019] The pharmacological effects of each Chinese herbal raw material in the fermented Chinese herbal preparation of this invention are as follows:

[0020] Gentian root: bitter in taste, cold in nature, enters the lung and liver meridians; has the effects of clearing heat and resolving phlegm, relieving cough and asthma, detoxifying and promoting diuresis, cooling blood and stopping bleeding; used for lung heat cough, excessive phlegm and wheezing, sore throat, canker sores, damp-heat jaundice, epistaxis, hematochezia, metrorrhagia, and external bleeding.

[0021] Artemisia capillaris: It tastes bitter and pungent, and is slightly cold in nature. It enters the spleen, stomach, liver, and gallbladder meridians. It has the effects of clearing heat and promoting diuresis, and relieving jaundice. It is used for damp-heat jaundice, difficulty in urination, infectious jaundice hepatitis, and wind-itchy sores and scabies.

[0022] Sedum sarmentosum: It has a sweet and bland taste, and is cool in nature. It enters the liver, gallbladder, and small intestine meridians. It has the effects of promoting diuresis and relieving jaundice, clearing heat and detoxifying. It is used for damp-heat jaundice, difficulty in urination, carbuncles and sores.

[0023] Lobelia chinensis: It has a pungent taste and a neutral nature, and enters the heart, small intestine, and lung meridians; it has the effects of clearing heat and detoxifying, promoting diuresis and reducing swelling; it is used for carbuncles, boils, snake and insect bites, ascites and edema, damp-heat jaundice, eczema and damp sores.

[0024] As a medicine: It tastes bitter and is cold in nature. It enters the liver, stomach, and large intestine meridians. It has the effects of clearing damp heat and strengthening the stomach. It is used for damp-heat jaundice, hypochondriac pain, dysentery with abdominal pain, and loss of appetite.

[0025] Dendrobium: It tastes sweet and is slightly cold in nature. It enters the stomach and kidney meridians. It has the effects of nourishing the stomach and promoting the production of body fluids, nourishing yin and clearing heat. It is used for febrile diseases with depletion of body fluids, dry mouth and thirst, insufficient stomach yin, poor appetite and dry retching, persistent low fever after illness, yin deficiency and fire excess, bone steaming fever, blurred vision, and weakness of muscles and bones.

[0026] White peony root: bitter and sour in taste, slightly cold in nature, and enters the liver and spleen meridians; it has the effects of nourishing blood and regulating menstruation, astringing yin and stopping sweating, softening the liver and relieving pain, and calming liver yang; it is used for blood deficiency and chlorosis, irregular menstruation, spontaneous sweating, night sweats, hypochondriac pain, abdominal pain, limb spasms, headache and dizziness.

[0027] Gynostemma pentaphyllum: bitter and slightly sweet in taste, cool in nature, and enters the lung, spleen, and kidney meridians; it has the effects of clearing heat and detoxifying, and tonifying deficiency; it is used for physical weakness and fatigue, seminal emission due to deficiency, leukopenia, hyperlipidemia, viral hepatitis, chronic gastroenteritis, and chronic bronchitis.

[0028] Goji berries: sweet in taste, neutral in nature, and enter the liver and kidney meridians; they have the effects of nourishing the liver and kidneys, benefiting essence and improving eyesight; they are used for deficiency of essence and blood, soreness of the waist and knees, dizziness and tinnitus, impotence and seminal emission, internal heat and thirst, blood deficiency and chlorosis, and blurred vision.

[0029] Polygonatum: sweet in taste, neutral in nature, enters the spleen, lung and kidney meridians; has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs and benefiting the kidneys; used for spleen and stomach qi deficiency, fatigue, stomach yin deficiency, dry mouth and poor appetite, lung deficiency and dry cough, consumptive cough with hemoptysis, deficiency of essence and blood, soreness and weakness of waist and knees, premature graying of hair, internal heat and thirst.

[0030] (III) Beneficial Effects

[0031] This invention provides a fermented traditional Chinese medicine preparation for liver protection and choleretic purposes in aquatic animals, and a method for its preparation. The fermented traditional Chinese medicine preparation is made from ten traditional Chinese medicines: gentian root, artemisia capillaris, sedum sarmentosum, lobelia chinensis, angelica sinensis, dendrobium, white peony root, gynostemma pentaphyllum, wolfberry, and polygonatum sibiricum. Through scientific formulation, these herbs exert a synergistic effect. At the same time, alkaline protease is added to promote digestion and absorption, reduce the burden on the liver, and brewer's yeast and Bacillus licheniformis are added to improve the intestinal microecological environment.

[0032] In vitro hepatocyte protection assay results show that the traditional Chinese medicine raw materials in the fermented traditional Chinese medicine preparation of the present invention can reduce the content of ALT and AST in the supernatant of HepG2 cells and the content of MDA in the cell lysate, and increase the activity of SOD and the level of T-AOC, thereby inhibiting the TGF-β-induced oxidative stress response, effectively restoring the function of the hepatocyte antioxidant defense system, and exerting a hepatoprotective effect.

[0033] This invention optimizes the processing of traditional Chinese medicine raw materials through synergistic fermentation technology, significantly improving the bioavailability of active ingredients and promoting effective absorption by the body. Simultaneously, it maintains intestinal microecological balance, further enhancing its comprehensive hepatoprotective and choleretic effects. In vivo experiments on spotted sea bass show that the fermented traditional Chinese medicine preparation of this invention can significantly reduce the levels of ALT, AST, TBA, and TBIL in the serum of spotted sea bass, while increasing SOD activity and the levels of CAT, GSH-Px, and T-AOC in liver tissue homogenate, and decreasing MDA content. This effectively alleviates D-GalN / LPS-induced hepatic lipid peroxidation. Furthermore, the fermented traditional Chinese medicine preparation of this invention can also promote bile secretion and reduce cholestasis, providing a fermented traditional Chinese medicine preparation with both hepatoprotective and choleretic effects for aquaculture. Attached Figure Description

[0034] Figure 1 For comparison of cell viability in each group.

[0035] Figure 2 Comparison of bile flow rates in different groups of spotted bass; Note: Compared with the blank control group, # P<0.05, ## P<0.01, compared with the model control group, * P<0.05, ** P<0.01. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] A fermented traditional Chinese medicine preparation for liver protection and choleretic purposes in aquatic animals, comprising the following parts by weight of raw materials: 25 parts gentian root, 22 parts artemisia capillaris, 20 parts sedum sarmentosum, 18 parts lobelia chinensis, 15 parts angelica sinensis, 12 parts dendrobium, 10 parts white peony root, 8 parts gynostemma pentaphyllum, 10 parts wolfberry, and 12 parts polygonatum sibiricum. In addition, it includes 20% alkaline protease, 22% brewer's yeast extract, and 10% Bacillus licheniformis extract, by weight of the raw materials.

[0039] The preparation method of the fermented traditional Chinese medicine preparation includes the following steps:

[0040] (1) Weigh each Chinese herbal raw material according to the above weight proportions, wash and dry it, pulverize it and pass it through a 100-mesh sieve, mix the obtained powder with 4-8 times the weight of deionized water, and stir to form a homogeneous suspension.

[0041] (2) Add alkaline protease to the suspension, adjust the pH to 6.5-7.5, and carry out the enzymatic hydrolysis reaction at 40-45℃ for 4-6 h. After the reaction is completed, filter and collect the hydrolysate.

[0042] (3) Activation of the strains: Saccharomyces cerevisiae was cultured in malt extract liquid medium at 30°C with shaking for 24 h, and Bacillus licheniformis was cultured in LB liquid medium at 37°C with shaking for 12 h to obtain Saccharomyces cerevisiae and Bacillus licheniformis liquid cultures, with a strain concentration of ≥1×10⁻⁶. 8 CFU / mL;

[0043] (4) Add 2wt% glucose and 1.5wt% peptone to the enzymatic hydrolysate as fermentation substrate, inoculate with Saccharomyces cerevisiae liquid and Bacillus licheniformis liquid according to the above weight ratio, control the fermentation temperature at 30-35℃, maintain the pH value at 5.5-6.5, carry out anaerobic / microaerobic fermentation for 5-7 days, filter the fermentation liquid, concentrate under reduced pressure, and then spray dry and granulate to obtain the fermented Chinese medicine preparation of the present invention.

[0044] Example 2

[0045] The difference between this embodiment and Embodiment 1 is that the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 15 parts of gentian root, 12 parts of artemisia capillaris, 10 parts of sedum sarmentosum, 8 parts of lobelia chinensis, 5 parts of angelica sinensis, 3 parts of dendrobium, 3 parts of white peony root, 3 parts of gynostemma pentaphyllum, 3 parts of wolfberry, and 4 parts of polygonatum sibiricum. In addition, it also includes 15% alkaline protease, 15% brewer's yeast liquid, and 3% Bacillus licheniformis liquid, accounting for 15% of the weight of the traditional Chinese medicine raw materials.

[0046] Example 3

[0047] The difference between this embodiment and Embodiment 1 is that the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 22 parts of gentian root, 19 parts of artemisia capillaris, 18 parts of sedum sarmentosum, 15 parts of lobelia chinensis, 12 parts of angelica sinensis, 8 parts of dendrobium, 7 parts of white peony root, 7 parts of gynostemma pentaphyllum, 7 parts of wolfberry, and 9 parts of polygonatum sibiricum. In addition, it also includes 18% alkaline protease, 20% brewer's yeast liquid, and 10% Bacillus licheniformis liquid, accounting for 18% of the weight of the traditional Chinese medicine raw materials.

[0048] Example 4

[0049] The difference between this embodiment and Embodiment 1 is that the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 17 parts of gentian root, 14 parts of artemisia capillaris, 12 parts of sedum sarmentosum, 10 parts of lobelia chinensis, 7 parts of angelica sinensis, 4 parts of dendrobium, 3 parts of white peony root, 3 parts of gynostemma pentaphyllum, 3 parts of wolfberry, and 4 parts of polygonatum sibiricum. In addition, it also includes 17% alkaline protease, 17% brewer's yeast liquid, and 8% Bacillus licheniformis liquid, accounting for 17% of the weight of the traditional Chinese medicine raw materials.

[0050] Example 5

[0051] The difference between this embodiment and Embodiment 1 is that the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 27 parts of Gentiana scabra, 24 parts of Artemisia capillaris, 22 parts of Sedum sarmentosum, 20 parts of Lobelia chinensis, 18 parts of Angelica sinensis, 15 parts of Dendrobium nobile, 13 parts of Paeonia lactiflora, 12 parts of Gynostemma pentaphyllum, 14 parts of Lycium barbarum, and 15 parts of Polygonatum sibiricum. In addition, it also includes 22% alkaline protease, 22% Saccharomyces cerevisiae culture, and 11% Bacillus licheniformis culture, accounting for 22% of the weight of the traditional Chinese medicine raw materials.

[0052] Example 6

[0053] The difference between this embodiment and Embodiment 1 is that the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 20 parts of Gentiana scabra, 17 parts of Artemisia capillaris, 15 parts of Sedum sarmentosum, 12 parts of Lobelia chinensis, 10 parts of Angelica dahurica, 6 parts of Dendrobium nobile, 5 parts of Paeonia lactiflora, 5 parts of Gynostemma pentaphyllum, 5 parts of Lycium barbarum, and 7 parts of Polygonatum sibiricum. In addition, it also includes 18% alkaline protease, 19% Saccharomyces cerevisiae culture, and 9% Bacillus licheniformis culture, accounting for 18% of the weight of the traditional Chinese medicine raw materials.

[0054] Example 7

[0055] The difference between this embodiment and Embodiment 1 is that the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 35 parts of Gentiana scabra, 32 parts of Artemisia capillaris, 30 parts of Sedum sarmentosum, 28 parts of Lobelia chinensis, 25 parts of Angelica dahurica, 22 parts of Dendrobium nobile, 20 parts of Paeonia lactiflora, 18 parts of Gynostemma pentaphyllum, 20 parts of Lycium barbarum, and 22 parts of Polygonatum sibiricum. In addition, it also includes 25% alkaline protease, 30% Saccharomyces cerevisiae culture, and 15% Bacillus licheniformis culture, accounting for 25% of the weight of the traditional Chinese medicine raw materials.

[0056] Example 8

[0057] The difference between this embodiment and Embodiment 1 is that the fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 30 parts of Gentiana scabra, 27 parts of Artemisia capillaris, 25 parts of Sedum sarmentosum, 23 parts of Lobelia chinensis, 20 parts of Angelica dahurica, 18 parts of Dendrobium nobile, 15 parts of Paeonia lactiflora, 16 parts of Gynostemma pentaphyllum, 15 parts of Lycium barbarum, and 18 parts of Polygonatum sibiricum. In addition, it also includes 23% alkaline protease, 26% Saccharomyces cerevisiae culture, and 13% Bacillus licheniformis culture, accounting for 23% of the weight of the traditional Chinese medicine raw materials.

[0058] Experimental Example 1

[0059] 1. Materials and Methods

[0060] 1.1 Test Drug

[0061] Experimental Group 1: Gentian root 25 g, Artemisia capillaris 22 g, Sedum sarmentosum 20 g, Lobelia chinensis 18 g, Angelica dahurica 15 g, Dendrobium nobile 12 g, Paeonia lactiflora 10 g, Gynostemma pentaphyllum 8 g, Lycium barbarum 10 g, Polygonatum sibiricum 12 g.

[0062] Experimental Group 2: Lobelia chinensis 18 g, Angelica sinensis 15 g, Dendrobium nobile 12 g, Paeonia lactiflora 10 g, Gynostemma pentaphyllum 8 g, Lycium barbarum 10 g, Polygonatum sibiricum 12 g.

[0063] Experimental Group 3: Gentian root 25 g, Artemisia capillaris 22 g, Sedum sarmentosum 20 g, Gynostemma pentaphyllum 8 g, Lycium barbarum 10 g, Polygonatum sibiricum 12 g.

[0064] Experimental Group 4: Gentian root 25 g, Artemisia capillaris 22 g, Sedum sarmentosum 20 g, Lobelia chinensis 18 g, Angelica sinensis 15 g, Dendrobium nobile 12 g, Paeonia lactiflora 10 g.

[0065] Weigh each Chinese herbal raw material according to the above weight ratio, wash, dry, pulverize and pass through a 100-mesh sieve. Mix the obtained powder with 6 times the weight of deionized water, extract by ultrasound for 30 min, and filter. Add 4 times the weight of deionized water to the filter residue again, sonicate for 30 min, filter, combine the two filtrates, and concentrate under reduced pressure to a drug mass concentration of 1 g / mL to obtain the Chinese herbal raw liquid of test groups 1-4.

[0066] 1.2 Cell Culture

[0067] HepG2 cells were revived in DMEM complete medium (hereinafter referred to as "complete medium") containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin dual antibiotics, and cultured routinely in a constant temperature incubator at 37°C and 5% CO2. Depending on the cell growth status, cells were passaged every 2-3 days using trypsin digestion, and cells in the logarithmic growth phase were used for experiments.

[0068] 1.3 Cell viability assay

[0069] HepG2 cells in the logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 96-well plates, with a blank control group and experimental groups 1-4, each with 3 replicates. Cells were incubated adherently for 24 h. The control group received 200 µL of complete medium, while experimental groups 1-4 received 200 µL of complete medium containing the drug (the final concentration of the herbal extract was 200 µg / mL). After 24 h of incubation, the medium was changed, adding 180 µL of medium and 20 µL of CCK-8 to each well. Cells were incubated for 1-4 h, and the absorbance was measured at 450 nm using a microplate reader to calculate cell viability. Cell viability = (OD200 + OD200) / (Cell Viability ... 药物组 -OD 空白组 ) / OD 空白组 ×100%.

[0070] 1.4 Protective effect against TGF-β-induced cell damage

[0071] HepG2 cells in the logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in 96-well plates, with a blank control group and experimental groups 1-4, each with 3 replicates, and cultured adherently for 24 h. Except for the blank control group, 50 µL of TGF-β solution (final TGF-β concentration of 10 ng / mL) was added to each of the other groups to establish a cell damage model. 150 µL of complete culture medium containing the drug (final concentration of the original Chinese medicine solution of 200 µg / mL) was added to each of experimental groups 1-4, while the blank control group received an equal volume of complete culture medium. Cells were cultured at 37℃ and 5% CO2 for another 24 h, and then centrifuged at 4℃ and 1000 rpm for 5 min. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the supernatant, and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) in the cell lysate were measured according to the ELISA kit instructions.

[0072] 1.5 Statistical Methods

[0073] All data were processed using Graphpad Prism 8.0 software. Data are expressed as mean ± standard deviation. ANOVA was used for analysis of variance when all groups were normally distributed. LSD-test was used for comparisons between groups with homogeneous variances. Dunnett's test was used for comparisons between groups with unequal variances. A p-value < 0.05 was considered statistically significant.

[0074] 2 Results

[0075] 2.1 Effects of each drug group on cell viability

[0076] Depend on Figure 1 It can be seen that, compared with the blank control group, there was no significant difference in cell viability in experimental groups 1-4, indicating that the raw materials of fermented Chinese medicine preparations did not show obvious cytotoxic effects and had good biological safety.

[0077] 2.2 Protective effect of each drug group against TGF-β-induced cell damage

[0078] Table 1 shows that, compared with the blank control group, the ALT and AST levels in the cell supernatant of the model control group were significantly increased (P<0.01), while the MDA content in the cell lysate was significantly increased (P<0.01), and the SOD activity and T-AOC level were significantly decreased (P<0.01), indicating that TGF-β successfully induced HepG2 cell damage. Compared with the model control group, the biochemical indicators of each treatment group showed an improvement trend. Among them, the ALT and AST levels in experimental group 1 decreased by 38.44% and 28.23%, respectively (P<0.01), and the MDA content in the cell lysate decreased by 33.17% (P<0.01), while the SOD activity and T-AOC level increased by 1.9 times and 1.7 times, respectively (P<0.01). These results indicate that the traditional Chinese medicine raw materials in the fermented traditional Chinese medicine preparation of this invention can effectively reverse TGF-β-induced oxidative stress damage in hepatocytes, thereby exerting a hepatoprotective effect.

[0079] Table 1 Comparison of biochemical indicators among the drug administration groups

[0080] Grouping ALT (U / L) AST (U / L) MDA (nmol / mg) SOD (U / mg) T-AOC (U / mg) Blank control group 7.59±1.63 8.41±0.88 23.25±3.14 82.70±2.61 5.19±0.82 Model control group 26.82 ± 1.45 ## ]] <![CDATA[18.88±1.45 ## ]]> <![CDATA[55.77±2.54 ## ]]> <![CDATA[34.40±1.94 ## ]]> <![CDATA[2.39±0.41 ## ]]> Experiment 1 group <![CDATA[16.51±0.94 ** ]]> <![CDATA[13.55±0.97 ** ]]> <![CDATA[37.27±1.86 ** ]]> <![CDATA[65.64±2.45 ** ]]> <![CDATA[4.03±0.48 ** ]]> Experimental Group 2 <![CDATA[19.87±1.44 ** ]]> <![CDATA[14.59±1.17 ** ]]> <![CDATA[45.68±2.23 ** ]]> <![CDATA[53.02±1.44 ** ]]> <![CDATA[3.71±0.41 * ]]> Experiment 3 groups <![CDATA[20.84±1.86 ** ]]> <![CDATA[15.35±0.49 ** ]]> <![CDATA[47.96±1.48 ** ]]> <![CDATA[46.82±1.88 ** ]]> 3.34±0.45 Experiment 4 groups 23.77±1.56 17.22±0.73 51.25±1.30 39.23±1.32 2.99±0.48

[0081] Note: Compared with the blank control group # P<0.05, ## P<0.01, compared with the model control group, * P<0.05, ** P<0.01.

[0082] 3. Conclusion

[0083] The herbal raw materials in the fermented traditional Chinese medicine preparation of this invention can inhibit TGF-β-induced oxidative stress by reducing the ALT and AST content in HepG2 cell supernatant and the MDA content in cell lysate, while increasing SOD activity and T-AOC levels. This effectively restores the function of the hepatocellular antioxidant defense system, exerts hepatoprotective activity, and exhibits good biosafety. The hepatoprotective effect was most significant in group 1, while the hepatoprotective activity of groups 2-4 was weakened to varying degrees due to the lack of certain herbal components. These data indicate that there is a significant synergistic effect among the various herbal raw materials in the fermented traditional Chinese medicine preparation of this invention, which is crucial for exerting the hepatoprotective effect and can serve as a basis for further research on its compatibility with enzyme preparations and compound fermentation agents.

[0084] Experimental Example 2

[0085] 1. Materials and Methods

[0086] 1.1 Test Drug

[0087] Silymarin is produced by Tianjin Tasly Shengte Pharmaceutical Co., Ltd.

[0088] The fermented traditional Chinese medicine preparation of the present invention is prepared according to the method described in Example 1 above.

[0089] Unfermented Traditional Chinese Medicine Preparation: Gentian root 25 g, Artemisia capillaris 22 g, Sedum sarmentosum 20 g, Lobelia chinensis 18 g, Angelica dahurica 15 g, Dendrobium nobile 12 g, Paeonia lactiflora 10 g, Gynostemma pentaphyllum 8 g, Lycium barbarum 10 g, Polygonatum sibiricum 12 g. Weigh each herb according to the above weight ratio, wash, dry with hot air at 60℃, pulverize and pass through a 100-mesh sieve. Mix the obtained powder with 6 times its weight of deionized water, ultrasonically extract for 30 min, and filter. Add 4 times its weight of deionized water to the residue and ultrasonically extract again for 30 min, filter, combine the two filtrates, concentrate under reduced pressure, and spray dry to obtain the unfermented traditional Chinese medicine preparation.

[0090] 1.2 Experimental Design

[0091] One hundred and fifty juvenile spotted bass were temporarily housed in a 1000 L recirculating aquaculture system. After two weeks of acclimatization feeding with a basic diet, they were randomly assigned to 15 indoor freshwater recirculating aquaculture tanks of 150 L each. The experiment was divided into five groups: a blank control group, a model control group, a milk thistle group, an unfermented traditional Chinese medicine preparation group, and a fermented traditional Chinese medicine preparation group (hereinafter referred to as the "fermented traditional Chinese medicine preparation group"). Each group had three replicates, with ten fish per replicate. The unfermented traditional Chinese medicine preparation group was fed a basic diet supplemented with 4 g / kg of the unfermented traditional Chinese medicine preparation, the fermented traditional Chinese medicine preparation group was fed a basic diet supplemented with 4 g / kg of the fermented traditional Chinese medicine preparation of this invention, and the milk thistle group was fed a basic diet supplemented with 48 mg / kg of milk thistle. After seven consecutive days of feeding, except for the blank control group, all other groups were intraperitoneally injected with 200 mg / kg D-GalN and 10 μg / kg LPS to induce the model. The blank control group was intraperitoneally injected with an equal volume of physiological saline. Samples were collected and indicators were detected 6 hours after model initiation. During the rearing period, the water temperature should be 29-31℃, dissolved oxygen ≥6 mg / L, and pH 5.9-6.8. Feed the fish thoroughly in the morning and evening, ensuring the composition and nutritional components of the basic feed meet the requirements of GB / T 22919.3-2008 "Aquatic Compound Feed Part 3: Perch Compound Feed".

[0092] 1.3 Detection Indicators

[0093] 1.3.1 Serum biochemical indicators

[0094] After the experiment, the fish were fasted for 24 hours. Two fish were randomly selected from each replicate and anesthetized with eugenol. Blood was collected from the tail vein. After standing at 4°C for 30 minutes, the fish were centrifuged at 2500 rpm for 10 minutes. The supernatant was collected and the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), and total bilirubin (TBIL) levels were determined according to the kit instructions.

[0095] 1.3.2 Liver Indicators

[0096] After the experiment, two fish were randomly selected from each replicate, their livers were dissected, weighed, and homogenized with physiological saline at a concentration of 100 mg / mL on ice to form a liver tissue homogenate. The homogenate was then centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was collected, and the contents of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) in the supernatant were determined according to the kit instructions.

[0097] 1.3.3 Measurement of bile secretion

[0098] After the experiment, two fish were randomly selected from each replicate and anesthetized via tail vein injection of 3% (w / v) sodium pentobarbital solution (1 mL / kg body weight). Once the fish were deeply anesthetized, they were fixed to a dissection table, and the abdominal cavity was opened along the midline to fully expose the digestive system. The gallbladder and surrounding connective tissue were meticulously separated, and the cystic duct was double-ligated using sterile sutures. The common bile duct was then located at the beginning of the duodenum, and a heparinized PE-50 catheter (0.5 mm inner diameter) was inserted, secured using a proximal-distal double ligation technique. The other end of the catheter was then inserted into a pre-cooled brown EP tube to 4°C, and bile collection was started from the first appearance of bile in the catheter and continued for 30 minutes. The entire procedure was performed in darkness to avoid degradation of photosensitive components such as bile acids.

[0099] 1.4 Statistical Methods

[0100] All data were processed using Graphpad Prism 8.0 software. Data are expressed as mean ± standard deviation. ANOVA was used for analysis of variance when all groups were normally distributed. LSD-test was used for comparisons between groups with homogeneous variances. Dunnett's test was used for comparisons between groups with unequal variances. A p-value < 0.05 was considered statistically significant.

[0101] 2 Results

[0102] 2.1 Effects of each group of drugs on serum biochemical indicators of spotted bass

[0103] As shown in Table 2, compared with the blank control group, the serum ALT, AST, TBA and TBIL levels in the model control group of spotted bass were significantly increased (P<0.01); compared with the model control group, the serum ALT, AST, TBA and TBIL levels in each treatment group of spotted bass were significantly decreased (P<0.01), with the fermented traditional Chinese medicine preparation group showing the most significant decreasing trend, indicating that the fermented traditional Chinese medicine preparation of the present invention can effectively improve liver function indicators of spotted bass.

[0104] Table 2 Comparison of serum biochemical indicators of sea bass in different groups

[0105] Grouping ALT (U / L) AST (U / L) TBA (μmol / L) TBIL (μmol / L) Blank control group 34.40±2.30 15.44±1.24 20.65±2.67 3.16±0.69 Model control group <![CDATA[67.50±2.27 ## ]]> <![CDATA[36.25±1.45 ## ]]> <![CDATA[73.96±2.38 ## ]]> <![CDATA[16.30±0.63 ## ]]> Positive control group <![CDATA[50.60±1.77 ** ]]> <![CDATA[26.83±1.48 ** ]]> <![CDATA[49.34±2.75 ** ]]> <![CDATA[9.44±0.84 ** ]]> Unfermented Chinese medicine preparation group <![CDATA[48.28±2.37 ** ]]> <![CDATA[24.32±1.06 ** ]]> <![CDATA[45.55±3.25 ** ]]> <![CDATA[7.57±1.15 ** <!-- 7 -->]]> Fermented Traditional Chinese Medicine Preparations Group <![CDATA[42.81±2.29 ** ]]> <![CDATA[19.86±1.31 ** ]]> <![CDATA[33.08±2.71 ** ]]> <![CDATA[5.87±0.42 ** ]]>

[0106] Note: Compared with the blank control group # P<0.05, ## P<0.01, compared with the model control group, * P<0.05, ** P<0.01.

[0107] 2.2 Effects of each group of drugs on liver indicators of spotted bass

[0108] As shown in Table 3, compared with the blank control group, the SOD activity, CAT, GSH-Px, and T-AOC content in the liver tissue homogenate of sea bass in the model control group were significantly decreased, while the MDA content was significantly increased (P<0.01). Compared with the model control group, the SOD activity, CAT, GSH-Px, and T-AOC content in the liver tissue homogenate of sea bass in each treatment group were significantly increased (P<0.01), while the MDA content was significantly decreased (P<0.01). Among them, the fermented traditional Chinese medicine preparation group showed the most significant improvement in liver indicators, indicating that the fermented traditional Chinese medicine preparation of the present invention can inhibit D-GalN / LPS-induced liver lipid peroxidation and oxidative stress, thereby alleviating liver damage.

[0109] Table 3 Comparison of liver indicators among different groups of spotted sea bass

[0110] Grouping SOD (U / mg) CAT (U / mg) MDA (nmol / mg) GSH-Px (μmol / g) T-AOC (U / mg) Blank control group 187.03±10.16 65.36±2.94 3.39±0.73 184.09±8.69 56.02±1.57 Model control group <![CDATA[104.04±7.13 ## ]]> <![CDATA[24.29±2.01 ## ]]> <![CDATA[17.77±1.47 ## ]]> <![CDATA[75.11±11.02 ## ]]> <![CDATA[27.36±0.79 ## ]]> Positive control group <![CDATA[141.08±15.35 ** ]]> <![CDATA[43.06±2.96 ** ]]> <![CDATA[11.04±0.83 ** ]]> <![CDATA[127.32±8.82 ** ]]> <![CDATA[39.74±1.67 ** ]]> Unfermented Chinese medicine preparation group <![CDATA[146.80±9.25 ** ]]> <![CDATA[46.00±3.04 ** ]]> <![CDATA[9.76±0.81 ** ]]> <![CDATA[138.22±6.58 ** ]]> <![CDATA[43.29±2.17 ** ]]> Fermented Traditional Chinese Medicine Preparations Group <![CDATA[163.40±10.62 ** ]]> <![CDATA[55.69±3.11 ** ]]> <![CDATA[6.29±0.64 ** ]]> <![CDATA[161.34±7.11 ** ]]> <![CDATA[48.67±1.73 ** ]]>

[0111] Note: Compared with the blank control group # P<0.05, ## P<0.01, compared with the model control group, * P<0.05, ** P<0.01.

[0112] 2.3 Effects of each group of drugs on bile flow rate in spotted bass

[0113] Depend on Figure 2 It can be seen that, compared with the blank control group, the bile flow of the sea bass in the model control group was significantly reduced (P<0.01); compared with the model control group, the bile flow of the sea bass in each treatment group was significantly increased (P<0.01), among which the fermented Chinese medicine preparation group showed the most significant increasing trend, indicating that the fermented Chinese medicine preparation of the present invention can alleviate bile stasis caused by liver damage.

[0114] 3. Conclusion

[0115] The above experimental data show that the fermented traditional Chinese medicine preparation of this invention has significant hepatoprotective and choleretic activities. It can effectively reduce the levels of ALT, AST, TBA, and TBIL in the serum of sea bass, while increasing the activity of SOD and the levels of CAT, GSH-Px, and T-AOC in liver tissue homogenate, and reducing the level of MDA, thereby effectively alleviating hepatic lipid peroxidation induced by D-GalN / LPS. Furthermore, the fermented traditional Chinese medicine preparation of this invention can also promote bile secretion and reduce cholestasis. The hepatoprotective and choleretic activities of the fermented traditional Chinese medicine preparation group are significantly better than those of the unfermented traditional Chinese medicine preparation group, indicating that optimizing the processing of traditional Chinese medicine raw materials through synergistic fermentation technology can significantly improve the bioavailability of active ingredients, promote effective absorption by the body, maintain intestinal microecological balance, and further enhance its comprehensive hepatoprotective and choleretic effects. This invention provides a fermented traditional Chinese medicine preparation with both hepatoprotective and choleretic effects for the aquaculture field.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fermented traditional Chinese medicine preparation for protecting the liver and promoting bile secretion in aquatic animals, characterized in that, The fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 15-35 parts of gentian root, 12-32 parts of artemisia capillaris, 10-30 parts of sedum sarmentosum, 8-28 parts of lobelia chinensis, 5-25 parts of angelica sinensis, 3-22 parts of dendrobium, 3-20 parts of white peony root, 3-20 parts of gynostemma pentaphyllum, 3-20 parts of wolfberry, and 4-22 parts of polygonatum sibiricum.

2. The fermented traditional Chinese medicine preparation according to claim 1, characterized in that, The fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 20-30 parts of gentian root, 17-27 parts of artemisia capillaris, 15-25 parts of sedum sarmentosum, 13-23 parts of lobelia chinensis, 10-20 parts of angelica sinensis, 7-17 parts of dendrobium, 5-15 parts of white peony root, 5-15 parts of gynostemma pentaphyllum, 5-15 parts of wolfberry, and 7-17 parts of polygonatum sibiricum.

3. The fermented traditional Chinese medicine preparation according to claim 1, characterized in that, The fermented traditional Chinese medicine preparation is made from the following parts by weight of traditional Chinese medicine raw materials: 25 parts of gentian root, 22 parts of artemisia capillaris, 20 parts of sedum sarmentosum, 18 parts of lobelia chinensis, 15 parts of angelica sinensis, 12 parts of dendrobium, 10 parts of white peony root, 8 parts of gynostemma pentaphyllum, 10 parts of wolfberry, and 12 parts of polygonatum sibiricum.

4. The fermented traditional Chinese medicine preparation according to any one of claims 1 to 3, characterized in that, It also includes alkaline protease, accounting for 15%-25% of the weight of Chinese medicinal materials, 15%-30% of brewer's yeast liquid, and 3%-15% of Bacillus licheniformis liquid.

5. The fermented traditional Chinese medicine preparation according to any one of claims 1 to 3, characterized in that, It also includes alkaline protease, which accounts for 20% of the weight of the Chinese medicinal materials; 22% of brewer's yeast liquid; and 10% of Bacillus licheniformis liquid.

6. The method for preparing fermented traditional Chinese medicine preparations according to any one of claims 1 to 5, characterized in that, Prepare according to the following steps: (1) Weigh each Chinese herbal raw material according to the above weight proportions, wash and dry it, then pulverize it through a 100-mesh sieve. Mix the obtained powder with 4-8 times the weight of deionized water and stir to form a homogeneous suspension. (2) Add alkaline protease to the suspension, adjust the pH to 6.5-7.5, carry out the enzymatic hydrolysis reaction at 40-45℃ for 4-6 hours, filter after the reaction, and collect the enzymatic hydrolysate; (3) Activation of the strains: Saccharomyces cerevisiae was cultured in malt extract liquid medium at 30°C with shaking for 24 h, and Bacillus licheniformis was cultured in LB liquid medium at 37°C with shaking for 12 h to obtain Saccharomyces cerevisiae and Bacillus licheniformis liquid cultures, with a strain concentration of ≥1×10⁻⁶. 8 CFU / mL; (4) Add 2wt% glucose and 1.5wt% peptone to the enzymatic hydrolysate as fermentation substrate, inoculate with Saccharomyces cerevisiae liquid and Bacillus licheniformis liquid according to the above weight ratio, control the fermentation temperature at 30-35℃, maintain the pH value at 5.5-6.5, carry out anaerobic / microaerobic fermentation for 5-7 days, filter the fermentation liquid, concentrate under reduced pressure, and then spray dry and granulate to obtain the fermented Chinese medicine preparation of the present invention.

7. The use of the fermented traditional Chinese medicine preparation according to any one of claims 1 to 5 in the preparation of a hepatoprotective and choleretic drug for aquatic animals.

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