Use of beta, beta-dimethylacrylshikonin in prevention and control of non-bacterial enteritis of carnivorous fish

Abstract

This invention discloses the application of β,β-dimethylacrylshikonin in the prevention and control of nonbacterial enteritis in carnivorous fish. Through experiments, the applicant discovered that, under the condition of replacing 50% of fishmeal protein in the feed with soybean meal protein, the addition of shikonin compounds, shikonin alcohol extract, or shikonin ultrafine powder can effectively improve the symptoms of enteritis in pearl grouper, increase the average weight gain rate of pearl grouper, improve the antioxidant capacity of pearl grouper, improve intestinal tissue structure, reduce the transcription levels of pro-inflammatory factors IL1β and IL8 in the intestine, and simultaneously upregulate the transcription level of anti-inflammatory factor IL10. Therefore, shikonin compounds, shikonin alcohol extract, or shikonin ultrafine powder have significant effects on soybean meal-induced enteritis in fish and can be used to prepare drugs or feed additives for the treatment or prevention of soybean meal-induced enteritis in fish.

Description

Technical Field

[0001] This invention relates to the use of shikonin compounds, specifically to the application of β,β-dimethylacryloylshikonin in the prevention and control of nonbacterial enteritis in carnivorous fish. Background Technology

[0002] The fish gut not only actively participates in the body's immune response, metabolism, and various stress responses, but is also responsible for obtaining nutrients and energy from the outside world to ensure the fish's growth and health. However, risk factors such as oil oxidation, protein spoilage, and mycotoxins in feed ingredients or feed products, as well as anti-nutritional factors from plant ingredients, often lead to metabolic disorders and even organ damage in fish, resulting in decreased disease resistance.

[0003] Fishmeal is a high-quality feed protein source. However, global fishmeal production is limited by resources and the environment, and the supply has remained relatively stable. With the continuous expansion of aquaculture worldwide, the demand for fishmeal is constantly increasing, leading to a prominent supply-demand imbalance and rising prices. Utilizing plant protein sources to replace fishmeal has become one of the effective methods to alleviate the fishmeal shortage. Soybean meal protein is considered a good alternative to fishmeal due to its relatively balanced amino acid composition, high animal digestibility, good palatability, stable source, resistance to pathogen contamination, strong antioxidant properties, and good spoilage resistance. However, soybean meal (SBM) contains anti-nutritional factors (such as soy saponins, trypsin inhibitors, and antigenic proteins), which reduce the nutrient utilization rate of feed. High-proportion replacement of fishmeal often induces soybean meal-induced enteritis (SBMIE) and liver lesions in fish, thus reducing their growth performance and limiting its application in aquatic animal feed. Studies have shown that soybean meal can replace up to 60% of the feed for grass carp (a herbivorous fish), up to 45% for carp and catfish (omnivorous fish), and up to 10% for cobia (a carnivorous fish) without affecting their health. However, when the replacement rate is 20% or 30%, it significantly affects production indicators such as weight gain, feed conversion ratio, survival rate, and condition factor. Literature reports that carnivorous fish, including European sea bass, croaker, Brazilian golden sea bass, pearl grouper, large yellow croaker, and cobia (all from the order Perciformes), rainbow trout and Atlantic salmon (from the family Salmonidae), turbot and flounder (from the genus *Scophthalmus*), and yellow catfish (from the genus *Pelteobagrus*) (from the order Siluriformes), can all develop symptoms of SBMIE.

[0004] Lithospermum erythrorhizon is a perennial herb belonging to the Boraginaceae family. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a medium-grade medicinal herb, with its root used medicinally. The Boraginaceae family comprises 100 genera and 2000 species, of which 49 genera and 208 species are found in my country. The Chinese Pharmacopoeia specifies that three genera of plants are used medicinally as Lithospermum erythrorhizon, primarily the dried roots of *Arnebia euchroma* (Royle) Johnst., *Lithospermum erythrorhizon* Sieb. et Zucc., or *Arnebia guttata* Bunge. The main bioactive components of Lithospermum erythrorhizon are naphthoquinone compounds, including shikonin, deoxyshikonin, β,β-dimethylacryloylshikonin, and acetylshikonin. Modern pharmacological studies have shown that Lithospermum erythrorhizon has strong antibacterial, antitumor, antiviral, and anti-inflammatory effects. For example, Zou Qianqian et al. proposed that the hydroxynaphthoquinone extract of Lithospermum erythrorhizon has an inhibitory effect on the release of TNF-α and IL-1β induced by LPS (lipopolysaccharide) in AW264.7 cells, and can reduce the expression of TNF-α in the colon tissue of TNBS (parabensulfonic acid) induced by IBD rats (Zou Qianqian et al., Effect of hydroxynaphthoquinone extract of Lithospermum erythrorhizon on the expression of inflammatory cytokines, Chinese Traditional and Herbal Drugs, Vol. 45, No. 13, July 2014, pp. 1894-1898).

[0005] There are currently no reports of using lithospermum extract, lithospermum ultrafine powder, or lithospermum naphthoquinone compounds in drugs or feed additives for treating nonbacterial enteritis in fish or enteritis caused by soybean meal. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide the application of β,β-dimethylacrylshikonin in the prevention and control of nonbacterial enteritis in carnivorous fish.

[0007] The first technical solution of the present invention is the application of shikonin compounds in the preparation of drugs or feed additives for treating or preventing nonbacterial enteritis in fish, wherein the shikonin compounds are one or a combination of two or more of acetylshikonin, β,β-dimethylacryloylshikonin, deoxyshikonin, shikonin and isobutyryloylshikonin.

[0008] In the first technical solution described above, the fish are preferably carnivorous fish raised in freshwater or seawater, specifically perciformes, salmonids, or flatfish. The non-bacterial enteritis is foodborne enteritis, or non-bacterial enteritis induced by soybean saponins or soybean anti-nutritional factors.

[0009] The applicant found in the experiment that β,β-dimethylacrylshikonin and isobutyrylshikonin can more significantly reduce the transcriptional levels of pro-inflammatory factors IL1β and IL8 in the fish intestine and more significantly increase the transcriptional level of anti-inflammatory factor IL10 in the fish intestine. Therefore, the shikonin compounds are preferably β,β-dimethylacrylshikonin and / or isobutyrylshikonin.

[0010] The applicant's experimental results show that shikonin compounds, particularly β,β-dimethylacrylshikonin and isobutyrylshikonin, can effectively inhibit the expression of L1β and IL8 in the intestinal tissue of fish suffering from nonbacterial enteritis, and increase the expression level of IL10 in the intestinal tissue of fish suffering from nonbacterial enteritis, thereby improving or maintaining the homeostasis of intestinal structure and reversing the effects of anti-nutritional factors. Therefore, shikonin compounds can be used to prepare drugs or feed additives for the treatment or prevention of nonbacterial enteritis in fish.

[0011] In the first technical solution mentioned above, the administration method of the naphthoquinone compound is oral, and the addition amount is 0.1 to 0.2‰ of the total weight of the feed.

[0012] On the other hand, the present invention also provides the use of lithospermum erythrorhizon extract or lithospermum erythrorhizon ultrafine powder in the preparation of drugs or feed additives for treating or preventing fish enteritis caused by feed with a high proportion of soybean meal replacing fishmeal. Typically, feed with a high proportion of soybean meal replacing fishmeal refers to feed in which soybean meal protein replaces 40-60% of fishmeal protein.

[0013] In the second technical solution described above, the lithospermum erythrorhizon alcohol extract is prepared using plants of the Boraginaceae family (including but not limited to Xinjiang lithospermum (Arnebia euchroma (Royle) Johnst.), Lithospermum erythrorhizon Sieb. et Zucc.), or Inner Mongolia lithospermum (Arnebia guttata Bunge)) as raw materials, with low-carbon alcohols as solvents, and employing existing conventional extraction methods. Preferably, the lithospermum erythrorhizon alcohol extract is prepared by heating or reflux extraction using lithospermum erythrorhizon as raw material and low-carbon alcohols with a concentration of 80–100 v / v% as solvents, followed by solvent recovery of the resulting extract. The low-carbon alcohol can specifically be methanol and / or ethanol, more preferably ethanol. The concentration of the low-carbon alcohol is more preferably 90–95 v / v%. In the lithospermum erythrorhizon alcohol extract, the content of β,β-dimethylacryloylshikonin is typically about 5–25% of the alcohol extract. The ultrafine powder of Lithospermum involved is a fine powder obtained by ultrafine grinding of plants of the Boraginaceae family (selected as above), usually ultrafine grinding to a particle size of 50-100μm.

[0014] In the second technical solution described above, when the lithospermum erythrorhizon alcohol extract is used as a feed additive, its addition amount accounts for 0.1-0.2% of the total feed weight; when the lithospermum erythrorhizon ultrafine powder is used as a feed additive, its addition amount accounts for 1.5-3.5 wt% of the total feed weight, more preferably 2-3 wt% of the total feed weight. In specific applications, adjustments can be made based on the selected species of lithospermum, the content of naphthoquinone compounds or β,β-dimethylacryloylshikonin in the alcohol extract or ultrafine powder, the proportion of soybean meal replaced in the feed, and the content of soybean saponins.

[0015] In the second technical solution mentioned above, the fish are preferably carnivorous fish raised in freshwater or seawater, such as fish of the order Perciformes, Salmoniformes, or Pleuronectiformes. Specifically, they can be pearl grouper, large (small) yellow croaker, bigmouth bass, mandarin fish, etc. of the order Perciformes; rainbow trout, Atlantic salmon, etc. of the family Salmonidae of the order Salmoniformes; turbot, flounder, etc. of the genus Scopolae of the family Flouronectiformes.

[0016] The applicant's experiments revealed that using extruded soybean meal as raw material, and with soybean meal protein replacing 50% of the fishmeal protein in the feed, the addition of shikonin compounds, shikonin alcohol extract, or shikonin ultrafine powder can effectively improve the symptoms of enteritis in pearl grouper, increase the average weight gain rate of pearl grouper, enhance the antioxidant and immune capabilities of pearl grouper, improve intestinal tissue structure, reduce the transcription levels of pro-inflammatory factors IL1β and IL8 in the intestine, and simultaneously upregulate the transcription level of anti-inflammatory factor IL10. Therefore, shikonin compounds, shikonin alcohol extract, or shikonin ultrafine powder have significant effects on soybean meal-induced enteritis in fish and can be used to prepare drugs or feed additives for the treatment or prevention of enteritis in carnivorous fish caused by soybean meal. Attached Figure Description

[0017] Figure 1 A photo of the extract of comfrey.

[0018] Figure 2 Neutrophil migration and migration statistics were presented in zebrafish tail-amplification inflammation models induced by comfrey extract and copper sulfate-induced inflammation. (A) shows neutrophil migration in zebrafish observed under a fluorescence stereomicroscope after administration of DEX and comfrey extract, respectively; (B) shows the neutrophil count and statistics at the zebrafish tail wound site; (C) shows the neutrophil count and statistics in the lateral line nerve mound region of zebrafish. Different letters indicate significant differences, while the same letter indicates no significant difference (P < 0.05, n = 10).

[0019] Figure 3The results show the effects of five shikonin monomers on the mRNA expression levels of IL1β, IL8, and IL10 in zebrafish intestinal tissue. (A) shows the expression level of IL1β in zebrafish intestinal tissue after administration of DEX and ZC1, ZC2, ZC3, ZC4, and ZC5, respectively, using real-time quantitative PCR; (B) shows the expression level of IL8 in zebrafish intestinal tissue after administration of DEX and ZC1, ZC2, ZC3, ZC4, and ZC5, respectively, using real-time quantitative PCR; (C) shows the expression level of IL10 in zebrafish intestinal tissue after administration of DEX and ZC1, ZC2, ZC3, ZC4, and ZC5, respectively, using real-time quantitative PCR. Different letters indicate significant differences; the same letter indicates no significant difference (P < 0.05, n = 3).

[0020] Figure 4 The diagram shows the dose-response relationship between different concentrations of β,β-dimethylacrylshikonin and the mRNA expression levels of IL1β, IL8, and IL10 in zebrafish intestinal tissue. (A) shows the expression level of IL1β in zebrafish intestinal tissue detected by real-time quantitative PCR after administration of DEX and ZC3-1, ZC3-2, ZC3-3, ZC3-4, and ZC3-5, respectively; (B) shows the expression level of IL8 in zebrafish intestinal tissue detected by real-time quantitative PCR after administration of DEX and ZC3-1, ZC3-2, ZC3-3, ZC3-4, and ZC3-5, respectively; and (C) shows the expression level of IL10 in zebrafish intestinal tissue detected by real-time quantitative PCR after administration of DEX and ZC3-1, ZC3-2, ZC3-3, ZC3-4, and ZC3-5, respectively. Different letters indicate significant differences, while the same letters indicate no significant differences (P < 0.05, n = 3).

[0021] Figure 5 Images of zebrafish intestinal tissue sections. (A) shows the group fed a whole zebrafish meal diet; (B) shows the group fed a whole zebrafish meal diet plus 5‰ soybean saponins; (C) shows the group fed a whole zebrafish meal diet plus 5‰ soybean saponins plus 0.2% β,β-dimethylacryloylshikonin; a: intestinal villi. Scale bar: 100 μm.

[0022] Figure 6 Images of intestinal tissue sections from the FM, SBM, and ZC groups of pearl grouper.

[0023] Among them, A: fish meal group (FM); B: soybean meal group (SBM); C: purple gromwell group (ZC); *: inflammatory cell infiltration; a: lamina propria; b: goblet cells.

[0024] Figure 7This graph shows the transcriptional levels of pro-inflammatory factors IL1β and IL8, and anti-inflammatory factor IL10 in the intestinal tissue of pearl grouper. A: Transcriptional level of IL1β in the intestinal tissue of the FM, SBM, and ZC groups; B: Transcriptional level of IL8 in the intestinal tissue of the FM, SBM, and ZC groups; C: Transcriptional level of IL10 in the intestinal tissue of the FM, SBM, and ZC groups. Different letters on the bars indicate significant differences (P<0.05). Detailed Implementation

[0025] To better explain the technical solution of the present invention, the present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0026] Example 1: Preparation of in vitro active experimental drugs

[0027] Lithospermum erythrorhizon Sieb. et Zucc.: Weigh an appropriate amount of lithospermum erythrorhizon Sieb. et Zucc., pulverize it, and pass it through a 100-mesh sieve; collect the sieve residue, add 95 v / v ethanol equivalent to 3 times its weight, reflux extract 3 times, 4 hours each time, combine the extracts, and recover the solvent by rotary evaporation to obtain a deep purple-red, viscous lithospermum erythrorhizon ethanol extract (ZC, of ​​which the total naphthoquinone content is 1.9%). Figure 1 As shown. When using, dilute with dimethyl sulfoxide to the concentration to be used.

[0028] Copper sulfate (AR), Sinopharm Chemical Reagent Co., Ltd.

[0029] Dexamethasone: DEX, Dexamethasone Sodium Phosphate Injection, Sichuan Hengtong Animal Health Biotechnology Co., Ltd.

[0030] Soybean saponins: Extruded soybean meal (Guangxi Gangqing Oil Co., Ltd.) was extracted by hot reflux with 80 v / v% ethanol. After filtration, the solvent was recovered until there was no alcohol odor. After dilution with water, the extract was adsorbed using D101 macroporous resin, washed with water, eluted with alcohol, and the solvent was recovered. The extract was then freeze-dried (see reference: Diao Xiaoqin, Tian Xiaoju, Guan Haining, Xu Guihua, Ma Songyan, Bai Xiaobo. Study on determination of soybean saponins using oleanolic acid as standard. Grain and Oil Processing, 2009(11):53-55). The content of soybean saponins was determined.

[0031] Example 2: Establishment of a zebrafish inflammation model and drug activity experiment

[0032] 1. Experimental Methods

[0033] a. Establishment and administration of a zebrafish copper sulfate inflammation model

[0034] Establishment of a copper sulfate model in zebrafish: Transgenic zebrafish embryos (MPO-eGFP, National Zebrafish Resource Center) at 24 hpf were immersed in copper sulfate solution to construct a copper sulfate inflammation model through chemical damage. Ten zebrafish embryos were randomly selected from each group for anti-inflammatory activity screening.

[0035] (1) Blank control group: Zebrafish embryos were soaked in distilled water for 2 hours.

[0036] (2) Copper sulfate model group: Zebrafish embryos were immersed in a solution containing copper sulfate (13.3 uM) for 2 h.

[0037] (3) Positive drug control group: Zebrafish embryos were soaked in distilled water containing copper sulfate (13.3 uM) and dexamethasone (DEX, 90 ug / ml) for 2 h.

[0038] (4) Experimental drug group: Zebrafish embryos were soaked in distilled water containing copper sulfate solution (13.3 uM) and lithospermum erythrorhizon extract (1 mg / ml) for 2 h.

[0039] b. Establishment and drug administration of a zebrafish tail amputation inflammation model

[0040] Establishment of a zebrafish tail amputation model: Transgenic zebrafish embryos (MPO-eGFP, National Zebrafish Resource Center) born at 24 hours postpartum were anesthetized, and the tails of the zebrafish were transversely cut under a stereomicroscope using a scalpel. The embryos were then revived to construct a zebrafish tail amputation inflammation model. Ten zebrafish embryos were randomly selected from each group for anti-inflammatory activity screening.

[0041] (1) Blank control group: Zebrafish embryos were soaked in distilled water for 2 hours.

[0042] (2) Tail-cutting model group: The tail of the zebrafish embryo was cut horizontally and placed in distilled water for 2 hours.

[0043] (3) Positive drug control group: The tail of the zebrafish embryo was cut off and placed in distilled water containing dexamethasone (DEX) (90ug / ml) for 2 hours.

[0044] (4) Drug administration group: The tail of the zebrafish embryo was cut off and placed in distilled water containing the lithospermum extract (1 mg / ml) prepared in Example 1 for 2 hours.

[0045] 2. Real-time observation of neutrophil migration in zebrafish (MPO-eGFP) embryos

[0046] Neutrophils in zebrafish (MPO-eGFP) were excited with blue light (415–455 nm) to emit green fluorescence. Neutrophil migration was observed under a fluorescence microscope (Olympus IX73P2F), and images of fluorescent neutrophil migration were captured and recorded. Image-pro plus 6.0 software was used to digitize the zebrafish neutrophil fluorescence migration images. SPSS 26.0 software was used, and one-way ANOVA with post-hoc multiple tests was employed to test and process the experimental data. Experimental results are expressed as mean ± standard error (SEM), and P < 0.05 was considered statistically significant.

[0047] The results are as follows Figure 2 As shown in the figure. In the copper sulfate inflammation model experiment, compared with the blank group, zebrafish embryos were damaged in copper sulfate solution, and a large number of neutrophils migrated and accumulated in the lateral line thalamus region. Compared with the model group, the number of neutrophils accumulating in the lateral line thalamus region was significantly reduced in the positive drug DEX group and the drug administration group, indicating that DEX and Lithospermum erythrorhizon can inhibit the migration of neutrophils to the lateral line thalamus region. In the tail amputation inflammation model experiment, after the embryo's tail was transversely cut, a large number of neutrophils accumulated at the tail wound site. Compared with the model group, the number of neutrophils accumulating at the tail wound site was significantly reduced in the positive drug DEX group and the drug administration group. The applicant found through experiments that DEX and Lithospermum erythrorhizon can significantly inhibit the migration of neutrophils to the site of inflammation and have anti-inflammatory activity.

[0048] Example 3: Isolation, extraction and structural identification of monomeric compounds from Lithospermum erythrorhizon

[0049] This embodiment provides a method for the isolation, extraction, and structural identification of monomeric compounds from Lithospermum erythrorhizon, as detailed below:

[0050] After pulverizing Lithospermum erythrorhizon Sieb. et Zucc., naphthoquinone was extracted using the supercritical carbon dioxide method. The specific extraction conditions were as follows: extraction temperature 44.4℃, pressure 29.5 MPa, separation pressure 8 MPa, separation temperature 47.5℃, extraction time 156 min. After carbon dioxide was recovered from the extract, the resulting paste was separated by silica gel column chromatography and gel column chromatography, using petroleum ether-chloroform and petroleum ether-ethyl acetate as solvent systems. The extracts were then separated, purified and identified as deoxyshikonin (ZC1), acetylshikonin (ZC2), β,β-dimethylacryloylshikonin (ZC3), shikonin (ZC4) and isobutyryloylshikonin (C5). (References: Ai Kehui, Li Fengying, Li Yong, Wang Weibao, Wu Yurong, Study on naphthoquinone components and determination of shikonin content in Lithospermum erythrorhizon, Acta Botanica Sinica, 1989, 31(7): 549-553; I. Morimoto, T. Kishi, S. Ikegami. Naphthoquinone derivatives from Lithospermum erythrorhizon Sieb. et Zucc. Tetrahedron Letters, 1965, (52): 4737-4739). The chemical structure of the obtained compound is shown below:

[0051]

[0052] Example 4: Evaluation of the anti-inflammatory activity of shikonin naphthoquinone compounds

[0053] 1. Experimental Methods

[0054] 1) Zebrafish husbandry

[0055] Soybean saponins were added to the diet of wild zebrafish (type AB, National Zebrafish Resource Center) at a mass ratio of 5‰ to induce enteritis and obtain a non-bacterial inflammation model group (Hedrera MI, Galdames JA, Jimenez-Reyes MF, et al. Soybean meal induces intestinal inflammation in zebrafish larvae[J]. PLoS One, 2013, 8(7):e69983); DEX was added to the diet of the inflammation model group; and the experimental diet of the drug group was prepared by adding the isolated Lithospermum erythrorhizon monomer compound obtained in Example 3 to the diet of the model group according to the concentration in Table 1. Before the experiment, the zebrafish were temporarily held for 3 days and then randomly divided into 8 groups, with 3 replicates in each group and 10 fish in each replicate. They were fed twice a day (8:00, 17:00) until they were apparently satiated. The experiment was carried out in a zebrafish farming system for 2 weeks.

[0056] Table 1. Evaluation of anti-inflammatory activity in zebrafish: Feed and drug ratio table

[0057]

[0058] Fishmeal Group (FM): Using a zebrafish whole fishmeal diet ( Small fish food).

[0059] Nonbacterial enteritis model group (SBM): zebrafish whole fish meal diet with 5‰ soybean saponins added according to feed weight ratio.

[0060] Positive drug group: zebrafish whole fish meal diet was used, with 5‰ soybean saponins and 0.2‰ DEX (90ug / ml) added at the mass ratio.

[0061] Drug group: zebrafish whole fish meal diet was used, with 5‰ soybean saponins and the corresponding amount of monomeric compounds added according to Table 1 by mass ratio.

[0062] 2) Sample collection

[0063] After the breeding experiment, three fish were randomly selected from each group, dissected using sterile dissection tools, and the last third of the intestinal tissue was cut off. After rinsing with pre-cooled PBS and cleaning the mesentery and contents, the fish were immediately placed in RNase-free EP tubes and immersed in liquid nitrogen, and then stored at -80°C for later use.

[0064] 3) Analysis of the expression levels of pro-inflammatory factors IL1β and IL8 and anti-inflammatory factor IL10 in intestinal tissue mRNA

[0065] Real-time quantitative PCR: Total RNA was extracted from intestinal tissues of each group using TRIZOL RNA extraction reagent and reverse transcribed into cDNA using a kit (Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Pur ge), Suzhou Nearshore Protein Technology Co., Ltd.). Using β-actin gene as an internal control, the expression levels of IL1β, IL8, and IL10 in intestinal tissues of each group were detected by real-time quantitative PCR (qPCR kit: SYBR qPCR SuperMix Plus kit, Suzhou Nearshore Protein Technology Co., Ltd.). Using 2... -△△Ct The transcriptional levels of IL1β, IL8, and IL10 in the intestines of zebrafish in each group were calculated. The results were analyzed using one-way ANOVA with IBM SPSS Statistics 26.0 software to determine their significance (p<0.05 indicates significant difference).

[0066] 2. Experimental Results

[0067] Real-time quantitative PCR results are as follows Figure 3 As shown in the results, compared with the SBM and DEX groups, among the five naphthoquinone compounds, β,β-dimethacrylshikonin (ZC3) and isobutyrylshikonin (ZC5) significantly reduced the transcription levels of pro-inflammatory factors IL1β and IL8 in zebrafish intestines, with β,β-dimethacrylshikonin showing the most significant reduction. Acetylshikonin, β,β-dimethacrylshikonin, shikonin, and isobutyrylshikonin significantly increased the expression level of anti-inflammatory factor IL10 in zebrafish intestines, with activities comparable to the positive control drug dexamethasone, with β,β-dimethacrylshikonin and isobutyrylshikonin being particularly significant. The experiment revealed that among the five major naphthoquinone chemical components of Lithospermum erythrorhizon, β,β-dimethacrylshikonin can significantly downregulate the expression levels of pro-inflammatory factors while upregulating the expression of anti-inflammatory factors, indicating that it has the most significant anti-inflammatory activity, and its application effects can be further explored.

[0068] Example 5: Application of β,β-dimethacryloylshikonin as a feed additive in nonbacterial enteritis

[0069] 1. Experimental Methods

[0070] 1) Zebrafish husbandry

[0071] In the diet of wild zebrafish (type AB, National Zebrafish Resource Center), soybean saponins were added at a mass ratio of 5‰ to induce enteritis in zebrafish, resulting in a non-bacterial inflammation model group (Hedrera MI, Galdames JA, Jimenez-Reyes MF, et al. Soybean meal induces intestinal inflammation in zebrafish larvae[J]. PLoS One, 2013, 8(7):e69983); the positive control group (DEX) was fed with DEX; the drug group was fed with the concentration of the isolated Lithospermum erythrorhizon monomer compound obtained in Example 3 according to Table 2. Before the experiment, zebrafish were temporarily held for 3 days, and then randomly divided into 8 groups, with 3 replicates in each group and 10 fish in each replicate. They were fed twice a day (8:00, 17:00) until they were apparently satiated. The experiment was conducted in a zebrafish farming system for 2 weeks.

[0072] Table 2 Drug Addition Dosage Table

[0073]

[0074] Fishmeal Group (FM): Using a zebrafish whole fishmeal diet ( (Small fish feed).

[0075] Nonbacterial enteritis model group (SBM): zebrafish whole fish meal diet with 5‰ soybean saponins added according to feed weight ratio.

[0076] Positive drug group: zebrafish whole fish meal diet was used, with 5‰ soybean saponins and 0.2‰ DEX (90ug / ml) added at the mass ratio.

[0077] Drug group: Zebrafish whole fish meal diet was used, with 5‰ soybean saponins added by weight and the corresponding amount of drugs added according to Table 2.

[0078] 2) Sample collection

[0079] Same as Example 4.

[0080] 3) The expression levels of pro-inflammatory factors IL1β and IL8 and anti-inflammatory factor IL10 in intestinal tissue were the same as in Example 4.

[0081] 2. Experimental Results

[0082] Real-time quantitative PCR results are as follows Figure 4As shown in the results, compared with the FM group, soy saponins significantly induced enteritis in zebrafish. Specifically, soy saponins significantly increased the mRNA expression levels of IL1β and IL8 in zebrafish intestinal tissue, while significantly decreasing the mRNA expression level of IL10. Compared with the SBM group, after administration, the mRNA expression levels of IL1β and IL8 in zebrafish intestinal tissue decreased with increasing concentrations of β,β-dimethacrylshikonin (0.05%, 0.1%, and 0.2%), while the mRNA expression level of IL10 increased with increasing concentrations of β,β-dimethacrylshikonin (0.05%, 0.1%, and 0.2%). The applicant found through experiments that β,β-dimethacrylshikonin can effectively inhibit the expression of IL1β and IL8 while promoting the expression of IL10, and this effect is dose-dependent within a certain concentration range (0.05%, 0.1%, and 0.2%).

[0083] Example 6: Effects of β,β-dimethylacryloylshikonin on the intestinal tissue structure of zebrafish

[0084] 1. Experimental Methods

[0085] 1) Zebrafish farming

[0086] Healthy zebrafish of similar size were randomly divided into three groups: fishmeal group (FM), soybean meal group (SBM), and drug group (ZC3). They were fed with the corresponding feeds in the zebrafish farming system for 2 weeks, with the daily feeding amount and farming environment the same as in Example 5.

[0087] Fishmeal Group (FM): Zebrafish whole fishmeal diet.

[0088] Soybean meal group (SBM): Zebrafish whole fish meal diet with 5‰ soybean saponins added according to feed weight ratio.

[0089] The treatment group (ZC3) was fed a zebrafish whole fish meal diet with 5‰ soybean saponins and 0.2% β,β-dimethylacrylol shikonin added at the same feed weight ratio.

[0090] 2) Sample collection

[0091] After the experiment, three fish were randomly selected from each group, and their intestines were aseptically collected and placed in 4% paraformaldehyde general tissue fixative to prepare intestinal tissue sections.

[0092] 3) HE section preparation

[0093] The fixed intestinal tissue was dehydrated with graded concentrations of ethanol (70–100 v / v%) and cleared with xylene. After embedding, sectioning, baking, and dewaxing, it was stained with hematoxylin and eosin sequentially. Finally, after dehydration, clearing, sealing, and drying, it was observed under a microscope.

[0094] 2. Experimental Results

[0095] Results of HE section of intestinal tissue Figure 5 As shown in the figure. The results showed that, compared with the FM group, the intestinal villus folds in the SBM group were significantly shortened, and the intestinal villus folds were lengthened after administration, indicating that β,β-dimethylacrylshikonin can improve the intestinal structure and restore intestinal function in fish.

[0096] The applicant discovered through experiments that shikonin naphthoquinone compounds, especially β,β-dimethylacryloylshikonin, can inhibit the occurrence of intestinal inflammation in fish, help restore intestinal structure and function, and improve non-bacterial enteritis in fish.

[0097] Example 7: Comparison of growth performance differences among groups of pearl grouper

[0098] Preparation of ultrafine powder of Lithospermum erythrorhizon: Weigh an appropriate amount of Lithospermum erythrorhizon Sieb. et Zucc., and ultrafine pulverize it to a particle size of 50 μm to obtain ultrafine powder of Lithospermum erythrorhizon (of which the total naphthoquinone compound content is 1.9%).

[0099] Instructions for use: Add ultrafine powder of Lithospermum erythrorhizon to the feed according to the mass ratio.

[0100] The feed formulations used in this experiment are shown in Table 3. The fishmeal group (FM) used a full fishmeal feed (steamed fishmeal, Corporación Pesquera Inca SAC, Bayovar Plant, Peru). The soybean meal group (SBM) used soybean meal protein to replace 50% of the fishmeal protein (expanded soybean meal, Guangxi Gangqing Oil Co., Ltd.). The comfrey group (ZC) used soybean meal protein to replace 50% of the fishmeal protein and added 3% comfrey ultrafine powder by weight of the feed. All three groups of feeds were equal in nitrogen (50% crude protein) and fat (10% crude fat). The content of naphthoquinones in the comfrey group feed was 0.5‰.

[0101] Referring to the feed formulation (Table 3), ultrafine powder of Lithospermum erythrorhizon was added to 5 kg of soybean meal protein, which replaced 50% of the fish meal protein, and mixed evenly. The mixture was then pelleted to obtain the experimental feed, namely the Lithospermum erythrorhizon (ZC) group. The total naphthoquinone content of this group was determined to be 0.5‰. The feed particle size was approximately 2 mm. The fish meal (FM) group feed and the soybean meal (SBM) group feed were prepared according to the same formulation (Table 3), producing 5 kg of feed each.

[0102] Table 3 Experimental Feed Formulation Table

[0103]

[0104]

[0105] a: Vitamin premix provides 2000mg of vitamin B1, 6000mg of vitamin B2, 1400mg of vitamin B6, 200mg of vitamin B12, 120000mg of vitamin C, 2000mg of biotin, 80000mg of calcium pantothenate, 2000mg of inositol, and 600mg of folic acid per kilogram of feed.

[0106] b: Mineral premixes provide 3000–12000 mg of copper, 5000–20000 mg of iron, 8000–32000 mg of zinc, 10000–40000 mg of manganese, 200–800 mg of iodine, and 100–400 mg of selenium per kg of feed. Lysine and methionine are used to regulate amino acid balance.

[0107] Two hundred and seventy pearl grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂) with an average weight of approximately (72.5±0.5) g were selected and temporarily held for one week. Before the experiment, they were anesthetized with MS-222 (ethyl m-aminobenzoate methanesulfonate, imported and packaged by Sigma-Aldrich) and then randomly divided into three groups: FM group, SBM group, and ZC group, with three replicates per group and 30 fish per replicate. Initial body weight and initial body length were measured. After the experiment began, they were fed the corresponding diets twice a day (8:00 and 17:00) until they reached apparent satiety level. Continuous aeration with air stones was used to maintain sufficient dissolved oxygen concentration. The average air temperature was 31℃. Fresh seawater was changed daily, with 60% of the water changed in the early stage and 100% in the later stage. The culture experiment lasted for 8 weeks. After the experiment, feeding was stopped, and 24 hours later, the fish were anesthetized using MS-222. Their final body weight and length were measured. Based on the measurement data, the average weight gain (AWGR), specific growth rate (SGR), and survival rate (SR) of the grouper were calculated. The calculation formulas are as follows:

[0108] Survival rate = (Number of survivors at the end / Number of survivors at the beginning) × 100%

[0109] Average weight gain rate = (final weight - initial weight) / initial weight × 100%

[0110] Specific growth rate = (ln final body weight - ln initial body weight) / number of feeding days × 100%

[0111] The results are shown in Table 4.

[0112] Table 4. Growth performance of pearl grouper in FM, SBM and ZC groups

[0113]

[0114] Different superscript letters in the same row indicate significant differences (p<0.05).

[0115] As shown in Table 4, no grouper died in the fishmeal, soybean meal, and alkanet groups, with a survival rate of 100%. The average weight gain and specific growth rate of the soybean meal group were significantly lower than those of the fishmeal and alkanet groups, while there were no significant differences in average weight gain and specific growth rate between the fishmeal and alkanet groups. The applicant's experiments revealed that adding soybean meal protein at a 50% ratio significantly affected the growth performance of pearl grouper. However, adding alkanet along with a high proportion of soybean meal significantly improved the growth performance of the grouper, achieving results similar to those of the fishmeal group. Therefore, alkanet exhibits a significant and long-lasting effect in improving soybean meal-induced enteritis in pearl grouper, with no recurrence.

[0116] Example 8: Comparison of intestinal antioxidant capacity among different groups of pearl grouper

[0117] Preparation of lithospermum erythrorhizon alcohol extract: Same as in Example 1.

[0118] Aquaculture experiment:

[0119] Referring to the feed formulation (Table 3), the lithospermum erythrorhizon extract was added to a 5 kg feed mixture (50% fishmeal replaced by soybean meal), mixed thoroughly, and then pelleted to obtain the experimental feed, namely the lithospermum erythrorhizon (ZC) group. The total naphthoquinone content in the lithospermum erythrorhizon group feed was determined to be 0.5‰. The feed particle size was approximately 2 mm. The fishmeal (FM) group feed and the soybean meal (SBM) group feed were prepared according to the same formulation (Table 3), producing 5 kg of feed each.

[0120] Two hundred and seventy pearl grouper (Epinephelus fuscoguttatus♀×E. lanceolatus♂) with an average weight of approximately (72.5±0.5) g were selected and temporarily held for one week. Before the experiment, they were anesthetized with MS-222 (ethyl m-aminobenzoate methanesulfonate, imported and packaged by Sigma-Aldrich) and then randomly divided into three groups: FM group, SBM group, and ZC group, with three replicates per group and 30 fish per replicate. Initial body weight and initial body length were measured. After the experiment began, they were fed the corresponding diets twice a day (8:00 and 17:00) until they reached apparent satiety level. Sufficient dissolved oxygen concentration was maintained using continuous aeration with air stones. The average air temperature was 31℃. Fresh seawater was changed daily, with 60% of the water changed in the early stage and 100% changed in the later stage. The culture experiment lasted for 8 weeks. After the experiment, 3 fish from each group were randomly selected, anesthetized with MS-222, and their intestines were aseptically collected to measure the levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in the intestines. The results are shown in Table 5.

[0121] Table 5. Intestinal enzyme activity of pearl grouper from FM, SBM and ZC groups

[0122]

[0123] Different superscript letters in the same row indicate significant differences (p<0.05).

[0124] As shown in Table 5, the activities of superoxide dismutase and catalase in the intestines of the soybean meal group were significantly lower than those in the fish meal and comfrey groups, while the malondialdehyde (MDA) activity was significantly higher in the soybean meal group than in the fish meal and comfrey groups. There was no significant difference in superoxide dismutase and MDA activities between the fish meal and comfrey groups. The applicant's experiments revealed that feeding a high proportion of soybean meal reduces the antioxidant capacity of the intestines of pearl grouper, while comfrey alcohol extract can significantly improve the antioxidant capacity of the intestines of pearl grouper induced by a high proportion of soybean meal, thereby preventing and treating soybean meal-induced enteritis.

[0125] Example 9: Observation of intestinal tissue structure in different groups of pearl grouper

[0126] The preparation, application, and aquaculture experiments of the comfrey extract were the same as in Example 8. After the experiment, three fish were randomly selected from each group, and their intestines were aseptically collected and preserved in 4% paraformaldehyde general tissue fixative for intestinal tissue sectioning. The fixed intestinal tissue was dehydrated with a series of ethanol (70-100% concentration), cleared with xylene, embedded, sectioned, baked, and dewaxed, then stained with hematoxylin and eosin sequentially. Finally, after dehydration, clearing, sealing, and drying, it was observed under a microscope. The results are as follows: Figure 6 As shown.

[0127] like Figure 6 The results showed that the intestinal villi folds of the grouper in the soybean meal group were significantly shorter than those in the fishmeal group, with a significant increase in inflammatory cell infiltration, a significantly wider lamina propria, and a significantly increased number of goblet cells. Compared with the soybean meal group, the group with *Lithospermum erythrorhizon* showed a significant decrease in inflammatory cell infiltration, a significantly narrower lamina propria, and a significantly reduced number of goblet cells. The applicant's experiments revealed that feeding high-soybean meal diets induces enteritis in pearl grouper, and *Lithospermum erythrorhizon* can effectively improve and repair the intestinal structure caused by soybean meal-induced enteritis.

[0128] Example 10: Comparison of the differences in the transcriptional levels of pro-inflammatory factors IL1β and IL8 and anti-inflammatory factor IL10 in the intestinal tissue of different groups of pearl grouper

[0129] The preparation method, usage method, and aquaculture experiment of the comfrey extract were the same as in Example 8. Further, after the experiment, three fish were randomly selected from each group, anesthetized with MS-222, and their intestines were aseptically collected and placed in RNase-free preservation tubes to detect the transcriptional levels of pro-inflammatory factors IL1β and IL8 and anti-inflammatory factor IL10 in the intestinal tissue of pearl grouper.

[0130] Further, total RNA was extracted from the intestines. Intestinal tissue stored at -80°C was used, and extraction was performed using Trizol reagent (Thermo Scientific) according to the instructions. Genomic DNA was removed using DNase I. RNA purity (OD260 / 280 ratio) was measured using Nanodrop 2000. The RNA was reverse transcribed into cDNA using a reverse transcription kit (Plus All-in-one 1st Strand cDNASynthesis SuperMix (gDNA Purge), Suzhou Nearshore Protein Technology Co., Ltd.), and stored at -80°C.

[0131] Further, the transcriptional levels of pro-inflammatory factors IL1β and IL8 and anti-inflammatory factor IL10 in intestinal tissue were determined. Using β-actin gene as an internal control, and IL1β, IL8, and IL10 as target genes, cDNA from the intestinal tissue of *Gentiana spp.* was used as a template. RT-qPCR was performed using a real-time PCR instrument, with each reaction repeated three times. After the reaction, the results were analyzed using QuantStudio™ 6Flex Real-Time PCR System software, and the Ct values ​​of three replicate melting curves with similar single peaks were selected. Using 2... -△△Ct The transcriptional levels of IL1β, IL8, and IL10 in the intestinal tissue of the pearl grouper were calculated. The results were then analyzed using a one-way ANOVA with IBM SPSS Statistics 26 software to determine their significance (p < 0.05 indicated significant difference). The results are as follows: Figure 7 As shown.

[0132] like Figure 7 The results showed that the transcription levels of pro-inflammatory factors IL1β and IL8 in the zebrafish intestinal tissue were significantly higher in the soybean meal group than in the fish meal group and in the Lithospermum erythrorhizon group, indicating that soybean meal clearly induced intestinal inflammation. The lower levels of pro-inflammatory factors in Lithospermum erythrorhizon compared to the soybean meal group indicated that it could inhibit pro-inflammatory factors in the grouper intestine and significantly improve intestinal health. Compared to the fish meal group, the transcription level of the anti-inflammatory factor IL10 was significantly downregulated in the soybean meal group, while the expression level of the anti-inflammatory factor was higher in the Lithospermum erythrorhizon group, demonstrating a strong effect in reversing the pro-inflammatory factors induced by soybean meal. The applicant's experiments found that Lithospermum erythrorhizon can effectively reduce the transcription levels of pro-inflammatory factors IL1β and IL8 in the intestinal tissue of pearl grouper fed a high-soybean meal diet, while increasing the transcription level of the anti-inflammatory factor IL10, further demonstrating that the alcoholic extract of Lithospermum erythrorhizon has a significant effect on preventing and treating intestinal inflammation induced by a high proportion of soybean meal in pearl grouper.

Claims

1. The application of naphthoquinone compounds in the preparation of drugs or feed additives for the treatment or prevention of nonbacterial enteritis in fish, among which, The aforementioned shikonin compound is β,β-dimethylacryloylshikonin; the aforementioned nonbacterial enteritis is nonbacterial enteritis caused by soybean saponins or soybean meal.

2. The application according to claim 1, characterized in that, The fish described are carnivorous.

3. The application according to claim 2, characterized in that, The fish mentioned are perch, salmonid, or platypus.

4. The application of lithospermum erythrorhizon extract or ultrafine lithospermum erythrorhizon powder in the preparation of drugs or feed additives for treating or preventing enteritis in fish caused by a high proportion of soybean meal replacing fishmeal; among which, The aforementioned lithospermum erythrorhizon alcohol extract is obtained by using lithospermum erythrorhizon as raw material and low-carbon alcohol with a volume concentration of 80-100% as solvent, and then recovering the solvent from the extract.

5. The application according to claim 4, characterized in that, When the lithospermum erythrorhizon extract is used as a feed additive, its addition amount accounts for 0.1~0.2% of the total feed weight; when the lithospermum erythrorhizon ultrafine powder is used as a feed additive, its addition amount accounts for 1.5~3.5wt% of the total feed weight.

6. The application according to claim 4 or 5, characterized in that, The fish mentioned are perch, salmonid, or platypus.

7. The application according to claim 4 or 5, characterized in that, The fish species mentioned are pearl grouper, large yellow croaker, small yellow croaker, bigmouth bass, rainbow trout, Atlantic salmon, turbot, or flounder.

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