A caragana condensed tannin preparation for repairing inflammatory damage to sheep small intestinal epithelial cells and an effective concentration screening method thereof

Abstract

The application discloses a caragana condensate tannin preparation for repairing inflammatory damage of sheep small intestinal epithelial cells and a screening method thereof. The active ingredient of the preparation is caragana condensate tannin with a purity of greater than or equal to 95%, which is extracted by 70% acetone, purified by D101 resin, and compounded with a carrier to form a powder with a tannin content of 5% to 10%; the optimal concentration in vitro is 40 μg / mL, and 0.1% is added to the daily diet of lambs. The screening method takes immortalized sheep small intestinal epithelial cells as a model, 1 μg / mL LPS is used for modeling, and after gradient preparation intervention, four indexes of cell activity, inflammation, antioxidant and tight junction gene are detected to comprehensively determine the effective concentration. The test shows that the preparation with a concentration of 40 μg / mL has the effects of anti-inflammation, anti-oxidation and repairing intestinal barrier and has no cytotoxicity; feeding can significantly improve the daily weight gain of lambs and reduce the feed conversion ratio. The preparation and the in vitro screening system have good repeatability, low cost and fast detection, solve the problems of unstable tannin purity, no standard dosage and the like, and are suitable for the development of sheep intestinal health feed additives.

Description

Technical Field

[0001] This invention belongs to the field of ruminant gut health and feed biotechnology, specifically relating to a condensed tannin preparation of *Caragana korshinskii* for repairing inflammatory damage to sheep small intestinal epithelial cells and a method for screening its effective concentration. Background Technology

[0002] In large-scale sheep farming, stress, high-concentrate feeding, and pathogenic bacterial infections can easily trigger intestinal inflammation and damage, which is a core issue leading to decreased growth performance, weakened immunity, and reduced economic benefits in sheep. Intestinal epithelial cells, as a core component of the sheep's intestinal barrier, play a crucial role in nutrient absorption, immune defense, and blocking the invasion of intestinal endotoxins. Lipopolysaccharide (LPS), a core toxic component of Gram-negative bacterial cell walls, is a typical inducing factor for oxidative stress, inflammatory infiltration, and barrier structure damage in ruminant intestinal epithelial cells. It can activate the TLR4 / NF-κB inflammatory signaling pathway, triggering a large release of inflammatory factors, disrupting the tight junction structure of the intestine, increasing intestinal permeability, and subsequently inducing a systemic inflammatory response, severely hindering the high-quality development of large-scale sheep farming.

[0003] Currently, the industry's methods for preventing and controlling enteritis in sheep still have significant shortcomings. Traditional prevention and control methods rely heavily on antibiotics and chemical anti-inflammatory drugs. Long-term use can easily lead to problems such as drug resistance residues in animals, intestinal flora imbalance, and substandard livestock product safety, which does not meet the requirements of green livestock farming development. Among existing green alternatives, various plant extracts have become a hot topic in feed additive research and development. Condensed tannins, as natural polyphenolic active substances, possess multiple biological activities such as anti-inflammatory, antioxidant, antibacterial, and intestinal barrier repair, showing good application potential in the regulation of intestinal health in livestock and poultry.

[0004] Caragana korshinskii, a widely cultivated shrub in the arid regions of Northwest my country, boasts abundant resources, high renewability, and low planting costs. Its stems and leaves are rich in highly active condensed tannins, making it a natural green feed additive with significant development potential. Current research, however, focuses only on the basic efficacy verification of crude tannin extracts from Caragana korshinskii, demonstrating its ability to improve the antioxidant capacity of lambs, alleviate liver inflammatory damage caused by exogenous stress, and enhance basic intestinal barrier function. Furthermore, existing experimental vectors are mostly mouse heterologous cell models or whole-body epithelial experiments on live lambs, failing to focus on the specific damage mechanisms of sheep small intestinal epithelial cells, indicating a significant technological gap and deficiency. On the one hand, existing technologies have not disclosed or confirmed the targeted repair effect of condensed tannins from Caragana korshinskii on LPS-induced specific inflammatory and oxidative complex damage to sheep small intestinal epithelial cells, and have not clarified its precise mechanism of action in anti-inflammatory, antioxidant, and barrier repair of sheep intestinal cells, resulting in a vague core technical mechanism. On the other hand, there is no dedicated and standardized concentration screening system for condensed tannins from Caragana korshinskii in the industry. The existing feed addition amounts are all based on experience in live feeding, without the support of quantitative data at the in vitro cellular level. This generally suffers from technical pain points such as poor concentration adaptability, ineffectiveness at low concentrations, vague effective concentration range, prominent cytotoxicity at high concentrations, and unstable repair effects, making it impossible to achieve precise application.

[0005] Meanwhile, traditional methods for screening the concentration of plant tannins in feed additives all employ live, graded-feeding trials in sheep. These methods suffer from inherent drawbacks such as long trial periods, significant individual animal variability, numerous environmental interferences, high costs, large errors in biochemical indicator detection, and poor data repeatability and stability. They cannot quickly and accurately quantify the optimal effective concentration for repairing small intestinal epithelial cell damage in sheep, thus hindering the long-term industrial application of *Caragana korshinskii* condensed tannins in the precise control of intestinal inflammation in sheep and as a specialized green feed additive. In summary, the industry currently lacks a standardized screening method for the effective concentration of *Caragana korshinskii* condensed tannins targeting LPS-induced damage to sheep small intestinal epithelial cells, and there is no precise concentration screening technology based on sheep homologous immortalized cell models. This is a core technical challenge that urgently needs to be addressed in this field. Summary of the Invention

[0006] Addressing the core technical pain points and gaps in existing technologies, such as the lack of specific application of condensed tannins from *Caragana korshinskii* for targeted repair of LPS-induced sheep small intestinal cell damage, unclear mechanism of action, lack of a precise homologous cell screening system, lack of quantitative standards for concentration parameters, low precision of traditional screening methods, and poor industrial applicability, this invention provides a condensed tannin preparation from *Caragana korshinskii* for repairing inflammatory damage to sheep small intestinal epithelial cells and a method for screening its effective concentration. It constructs a standardized, repeatable, and quantifiable method for screening effective concentrations based on a sheep homologous immortalized small intestinal epithelial cell model, precisely defining the optimal concentration range while balancing damage repair efficiency and cell biosafety.

[0007] To achieve the above-mentioned objectives, this invention provides a *Caragana korshinskii* condensed tannin preparation for repairing inflammatory damage to sheep small intestinal epithelial cells. The active ingredient is *Caragana korshinskii* condensed tannin with a purity ≥95%. The *Caragana korshinskii* condensed tannin is obtained by extracting from *Caragana korshinskii* stems and leaves with 70% acetone and purifying by gradient elution with D101 macroporous resin. The safe and effective concentration of the preparation for action on immortalized sheep small intestinal epithelial cells is 40 μg / mL. The addition ratio to the diet of Dorpern lambs is 0.1%.

[0008] Furthermore, the formulation is a powder, the excipient is corn starch / wheat bran carrier, and the mass fraction of tannin active ingredient is 5%~10%.

[0009] Furthermore, the purification process of condensed tannins from Caragana korshinskii involves a material-to-liquid ratio of 1:15, extraction at 50°C for 2 hours, followed by washing with D101 resin to remove impurities and elution with 70% acetone.

[0010] Using self-constructed immortalized sheep small intestinal epithelial cells as a screening model, an inflammatory injury model was constructed using LPS, and gradient formulation interventions were set up. The repair effect of the formulations was quantitatively evaluated through four-dimensional indicators: cell activity, inflammatory factors, antioxidants, and tight junction genes. The formulation concentration with the best repair effect and no cytotoxicity was quickly screened. It can be used for the development of new formulations and the quality control of batch products, and can significantly replace live animal experiments.

[0011] This invention also provides a condensed tannin preparation of *Caragana korshinskii* for repairing inflammatory damage to sheep small intestinal epithelial cells and a method for screening its effective concentration, comprising the following steps: (1) Immortalized sheep small intestinal epithelial cells were injected at a concentration of 1×10⁻⁶. 5 Inoculate at a density of 100 cells / mL into culture plates and incubate for 12 hours until fully adherent; (2) Except for the blank control group, Escherichia coli LPS with a final concentration of 1 μg / mL was added for 24 h to construct an inflammatory-oxidative complex injury model; (3) Discard the LPS medium and add a complete medium containing a gradient concentration of Caragana condensed tannin for 24 h. The gradient concentrations are 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL and 160 μg / mL, respectively. (4) Cell viability was detected by CCK-8 assay, inflammatory factors TNF-α and IL-6 and antioxidant indicators SOD, GSH-Px and MDA were detected by ELISA, and the expression of tight junction genes Occludin and ZO-1 was detected by qPCR. (5) Based on the detection results of step (4), the concentrations that significantly improve cell survival rate, reduce inflammatory factors, improve antioxidant capacity, upregulate tight junction gene expression and have no cytotoxicity are selected and determined as the optimal in vitro effective and safe concentrations.

[0012] Furthermore, the LPS mentioned in step (2) is a lipopolysaccharide derived from Escherichia coli O55:B5.

[0013] Furthermore, the inflammatory factors TNF-α and IL-6 mentioned in step (4) were quantitatively detected using a double-antibody sandwich ELISA method; the antioxidant indicators SOD activity, GSH-Px activity and MDA content were detected using an ELISA kit; and the relative expression levels of the tight junction genes Occludin and ZO-1 were detected using real-time quantitative qPCR.

[0014] Furthermore, the optimal in vitro effective safe concentration mentioned in step (5) is 40 μg / mL.

[0015] Furthermore, the immortalized sheep small intestinal epithelial cells were constructed through the following steps: Select jejunal tissue from healthy newborn lambs, peel off the serosa and muscle layers, rinse with PBS, and cut into 1mm pieces. 3 The fragments were digested with 0.25% trypsin-EDTA for 20-30 minutes, then filtered and centrifuged to obtain primary cells; The human telomerase reverse transcriptase hTERT gene was introduced using lentiviral transfection. After transfection, the cells were selected with 400 μg / mL G418 for 10-14 days, and single clones were selected for amplification and culture. After more than 30 passages, immortalized sheep small intestinal epithelial cell lines were obtained.

[0016] Furthermore, the extraction and purification process of the condensed tannins from *Caragana korshinskii* includes: using a 70% acetone aqueous solution at a material-to-liquid ratio of 1:15 (g:mL), extracting at 50℃ for 2 hours, and collecting the crude extract by centrifugation; using D101 macroporous adsorption resin, loading the sample at 2.0mL / min, eluting with ultrapure water at 3.0mL / min to remove impurities, and then eluting with a 70% acetone aqueous solution at a gradient rate of 1.5mL / min, collecting the eluent, concentrating under reduced pressure, and vacuum drying to obtain condensed tannins from *Caragana korshinskii* with a purity ≥95%.

[0017] Further, the specific operation of the CCK-8 method for detecting cell viability in step (4) is as follows: add 10 μL CCK-8 reagent to each well, incubate in the dark for 2 hours, detect the absorbance value at a wavelength of 450 nm, and calculate the cell viability.

[0018] The condensed tannins from Caragana korshinskii are natural polyphenols obtained from the stems and leaves of Caragana korshinskii through acetone extraction and purification with macroporous resin, with a purity of not less than 95%.

[0019] The method described is a screening and evaluation method for the efficacy and usage concentration of in vitro formulations for non-therapeutic purposes.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The tannin purity of the preparation of the present invention is stable at ≥95%, and irritating impurities are removed. Compared with crude extract, it has lower cytotoxicity and higher intestinal repair efficiency. The powder formulation has good flowability, is suitable for feed mills to mix and process, and is convenient to store and transport. The standardized addition dosage is clearly defined at both the cell and living organism levels, which solves the pain point of the industry lacking quantitative addition standards.

[0021] (2) The sheep homologous immortalized small intestinal epithelial cell model is used, which has strong experimental specificity; the four-dimensional indexes are used for joint evaluation, and the results are accurate; the standardized operation procedure ensures that the experimental repeatability is ≥95%; compared with live feeding screening, the cycle is shortened by more than 60% and the experimental cost is reduced by 70%.

[0022] (3) Achieving a complete industrial chain technology loop of "raw material purification → formulation preparation → in vitro efficacy screening and quality control → live animal breeding application" not only protects the terminal feed additive products, but also protects the supporting in vitro evaluation and screening processes. The patent protection scope is comprehensive and the industrialization value is high. Attached Figure Description

[0023] Figure 1 A morphological comparison of immortalized sheep small intestinal epithelial cells and primary sheep small intestinal epithelial cells (magnified 200 times).

[0024] Figure 2 Immunofluorescence staining image of immortalized sheep small intestinal epithelial cells (200x magnification).

[0025] Figure 3: Line graph showing the change in the survival rate of sheep small intestinal epithelial cells after treatment with different concentrations of condensed tannins from Caragana korshinskii (the line graph visually illustrates the pattern of initial increase followed by decrease in toxicity). Figure 4: Bar chart comparing the levels of TNF-α and IL-6 inflammatory factors in the cell supernatant of each group (double bars, reflecting the anti-inflammatory effect). Figure 5: Bar chart comparing SOD, GSH-Px activity and MDA content in each group (three antioxidant indicators) Figure 6: Bar chart of relative expression levels of Occludin and ZO-1 tight junction gene mRNA in each group (barrier repair indicator) Detailed Implementation

[0026] The present invention will be further described in detail and completely below with reference to specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0027] Example 1: Extraction, purification and purity determination of condensed tannins from *Caragana korshinskii* This embodiment is used to prepare the high-purity condensed tannins of *Caragana korshinskii* used in this invention.

[0028] 1.1 Test Materials Caragana twigs and leaves: Harvested from the Ordos Desert Ecological Zone in Inner Mongolia, using healthy, tender shoots and leaves from the current year, free from pests, diseases, and mold. Raw material pretreatment: After natural air drying, dry in a 45℃ constant temperature oven until constant weight, pulverize, and pass through a 40-mesh standard sieve. Collect the sieve-passing powder, seal it, and store it in a desiccator away from light for later use.

[0029] Reference standard: Condensed tannin (proanthocyanidin) standard, purity ≥99.5%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Test reagents: Acetone, concentrated sulfuric acid, vanillin, methanol (chromatographic grade), anhydrous ethanol, etc., all analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.; Purification medium: D101 macroporous adsorption resin, Tianjin Nankai University Resin Factory, pretreated and activated before use.

[0030] 1.2 Test Instruments High-performance liquid chromatograph (Agilent 1260, Agilent Technologies, USA), UV-Vis spectrophotometer (TU-1901, Beijing Purkinje General Chemicals), constant temperature magnetic stirring water bath, high-speed refrigerated centrifuge (8000 r / min), vacuum rotary evaporator, electric thermostatic drying oven, electronic analytical balance (accuracy 0.0001 g), circulating water vacuum pump, pH meter, etc.

[0031] 1.3 Raw material pretreatment process The pretreated *Caragana korshinskii* powder was placed in a desiccator for equilibration for 24 hours and then accurately weighed for later use. To remove surface dust and water-soluble impurities from the raw material, it was quickly rinsed once with anhydrous ethanol, drained, dried at a low temperature of 40°C, cooled to room temperature, and then sealed for storage.

[0032] 1.4 Acetone Extraction Process The optimal extraction process parameters are as follows: acetone volume fraction 70%, extraction temperature 50℃, extraction time 2h, and material-to-liquid ratio 1:15 (g:mL).

[0033] Extraction procedure: Accurately weigh 10.00g of pretreated *Caragana korshinskii* powder, add 150mL of 70% acetone aqueous solution at a material-to-liquid ratio of 1:15, place in a constant temperature magnetically stirred water bath, and extract at 50℃ for 2 hours with stirring speed of 300r / min. After extraction, centrifuge at 8000r / min for 15min (temperature 4℃), collect the supernatant, repeat the extraction once with the residue, and combine the two supernatants to obtain the crude extract of *Caragana korshinskii* condensed tannins.

[0034] 1.5 Purification process (purification with D101 macroporous resin) The purification method employed was static adsorption combined with dynamic gradient elution using D101 macroporous adsorption resin.

[0035] Resin pretreatment: Soak D101 macroporous resin in anhydrous ethanol for 24 hours to fully dissolve residual monomers, porogens and other impurities inside the resin; dynamically rinse with anhydrous ethanol until the effluent is clear when water is added, then rinse with ultrapure water until there is no ethanol odor; then soak in 5% hydrochloric acid solution for 4 hours, rinse with ultrapure water until neutral; then soak in 5% sodium hydroxide solution for 4 hours, rinse with ultrapure water until neutral, and drain for later use.

[0036] Sample loading and adsorption: The pretreated resin was wet-packed into a column (2.6cm × 60cm), with a column bed volume of 200mL. The combined crude extract was concentrated under reduced pressure (45℃, -0.08MPa) to remove acetone solvent, yielding a water-soluble concentrate. The pH of the concentrate was adjusted to 4.0. The sample was loaded at a constant flow rate of 2.0mL / min, and static adsorption was allowed for 30min after loading.

[0037] Impurity removal elution: After adsorption saturation, impurities are removed by dynamic elution with ultrapure water at a flow rate of 3.0 mL / min and a washing volume of 3 times the column bed volume (600 mL) until the effluent is clear and free of turbidity.

[0038] Gradient elution of target components: First, elute with 40% acetone aqueous solution at a volume twice the column bed volume to remove weakly adsorbed impurities; then, use 70% acetone aqueous solution as the optimal eluent at a flow rate of 1.5 mL / min and an eluent volume four times the column bed volume (800 mL). Collect the eluent in fractions, with each 100 mL fraction as one part. Screen the eluent fractions with high tannin content by colorimetric reaction and combine the effective eluents.

[0039] Concentration and drying: The combined effective eluents were placed in a rotary evaporator and concentrated under reduced pressure at 45°C and -0.08MPa to completely remove acetone solvent and water, resulting in a viscous tannin extract. The extract was placed in a vacuum drying oven and dried under vacuum at 50°C to constant weight. It was then ground and pulverized to obtain a light yellow pure powder of Litsea cubeba condensed tannins. The powder was sealed, protected from light, and stored at low temperature.

[0040] 1.6 Purity Testing Methods A dual detection system was adopted, consisting of vanillin-sulfuric acid colorimetric method and high performance liquid chromatography (HPLC).

[0041] Colorimetric method: Accurately weigh 0.0100 g of purified *Leonurus japonicus* tannin sample, dissolve in methanol and dilute to 100 mL to prepare the sample test solution. Pipette 1.0 mL of gradient standard solution and the test solution into stoppered test tubes, add 5 mL of 1% vanillin-sulfuric acid solution, shake well, and develop color in a 60℃ water bath for 15 min. Cool to room temperature, and measure the absorbance at 500 nm using a blank reagent as a reference. Standard curve regression equation: y = 0.0286x + 0.0032, R² = 0.9997.

[0042] HPLC detection conditions: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-water (v / v 40:60); flow rate: 1.0 mL / min; detection wavelength: 280 nm; column temperature: 30 ℃; injection volume: 20 μL; isocratic elution. Purity was calculated using the external standard method.

[0043] 1.7 Purity Test Results Three parallel tests were set up (sample numbers S1, S2, and S3), and the test results are as follows: Table 1. Results of condensed tannin purity tests in different batches of *Caragana korshinskii*

[0044] The results showed that the average purity of the condensed tannins obtained from Caragana korshinskii was 95.31%, with a purity ≥ 95% and a relative standard deviation (RSD) of 0.19%, indicating good process repeatability and strong stability.

[0045] Example 2: Construction of an immortalized sheep small intestinal epithelial cell line This embodiment is used to construct the immortalized sheep small intestinal epithelial cell line used in this invention.

[0046] 2.1 Isolation and Culture of Primary Sheep Small Intestinal Epithelial Cells Select healthy newborn lambs (within 24 hours of birth) and harvest approximately 10 cm of jejunal tissue under aseptic conditions. Rinse the intestinal contents repeatedly with pre-cooled sterile PBS buffer until the rinsing solution is clear. Place the intestinal tissue in a sterile culture dish, peel off the serosa and muscle layers, and cut the remaining mucosa into tissue fragments of approximately 1 mm³.

[0047] Add 0.25% trypsin-0.02% EDTA digestion solution and incubate at 37°C for 20-30 minutes, gently pipetting every 5 minutes. After digestion, add an equal volume of DMEM / F12 complete medium containing 10% fetal bovine serum to terminate the digestion. Filter the suspension through a 200-mesh sterile cell sieve, collect the filtrate, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cell pellet, and seed it into culture flasks. Incubate at 37°C, 5% CO2, and saturated humidity. After 24 hours, replace the medium with fresh medium to remove non-adherent cells. Change the medium every 24 hours thereafter until the cell confluence reaches 80%-90%, obtaining purified primary sheep small intestinal epithelial cells.

[0048] 2.2 Lentiviral-mediated hTERT gene transfection Second-generation primary cells were harvested and their density adjusted to 5 × 10⁻⁶. 5Inoculate lentiviruses at a concentration of 1 / mL into 6-well plates and culture until approximately 70% confluence. Discard the medium and add lentiviral solution (MOI=50) carrying the human telomerase reverse transcriptase (hTERT) gene, along with 8 μg / mL Polybrene transfection aid. Transfect at 37°C for 12 h. After transfection, replace with fresh complete medium and continue culturing for 48 h.

[0049] 2.3 Screening of positive cells and establishment of immortalized cell lines After transfection, cells were treated with G418 selection reagent at a final concentration of 400 μg / mL for 10-14 days. The culture medium containing G418 was replaced every 3 days, and untransfected negative cells were discarded. After selection, vigorous monoclonal positive cell lines were picked under a microscope and transferred to 48-well plates for expansion culture, gradually increasing the scale to culture flasks. After more than 30 passages of continuous passage, a stable, continuously passageable immortalized sheep small intestinal epithelial cell line was obtained.

[0050] 2.4 Immortality Verification (1) Cell morphology verification: Continuous culture and observation showed that immortalized sheep small intestinal epithelial cells all exhibited typical cobblestone-like morphology, with clear cell boundaries, neat arrangement, no fibrosis or atypia, uniform and stable morphology, and no senescence or shedding (e.g. Figure 1 As shown); while primary cells cultured to the 8th generation exhibited morphological shrinkage, fragmentation, and extensive apoptosis (as shown). Figure 1 (As shown).

[0051] (2) Verification of proliferation capacity: CCK-8 test results showed that the immortalized cells had a stable proliferation rate in continuous culture and the cell doubling time was stable at 22-24h; the primary cells proliferated normally in the first to sixth generations, the proliferation rate decreased by more than 35% in the seventh generation, and the proliferation capacity was basically lost in the eighth generation, proving that the cell line successfully achieved immortalization and broke through the proliferation limitations of the primary cells.

[0052] (3) Identification of epithelial origin: Immunofluorescence staining was performed using CK18 antibody. Immortalized cells showed strong positive expression (e.g., Figure 2 As shown in the figure, it is confirmed that it is indeed of small intestinal epithelial cell origin and there is no fibroblast contamination.

[0053] Example 3: Concentration screening test of condensed tannin preparations from *Leonurus japonicus* to repair LPS-induced damage to sheep small intestinal epithelial cells. 3.1 Test Materials Caragana condensed tannin: Caragana condensed tannin preparation: prepared by combining Caragana condensed tannin with corn starch carrier and a purity of 96.2% obtained from Example 1, with an active tannin content of 8%.

[0054] Immortalized sheep small intestinal epithelial cell line: constructed and preserved according to Example 2.

[0055] LPS: Escherichia coli O55:B5, Sigma.

[0056] Main reagents: DMEM / F12 complete culture medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody), 0.25% trypsin, CCK-8 kit, TNF-α and IL-6 ELISA kit, SOD / GSH-Px / MDA detection kit, TRIzol, SYBR Green qPCR premix.

[0057] Main instruments: CO2 constant temperature incubator, ultra-clean workbench, enzyme-linked immunosorbent assay (ELISA) reader, real-time fluorescence quantitative PCR instrument.

[0058] 3.2 Cell Culture and Plating Immortalized sheep small intestinal epithelial cells were revived and placed in an incubator at 37°C, 5% CO2, and saturated humidity, and cultured routinely in DMEM / F12 complete medium. The medium was changed every 24 hours, and cells were passaged when confluence reached 85%. Cells in the logarithmic growth phase were harvested, digested with 0.25% trypsin for 2 minutes, and the digestion was stopped by adding culture medium. The cells were centrifuged at 1000 rpm for 5 minutes, resuspended, and the cell density was adjusted to 1 × 10⁶ cells / min. 5 The cells / mL were seeded into 96-well plates (100 μL per well for CCK-8 and ELISA detection) and 6-well plates (2 mL per well for qPCR detection), and cultured for 12 h until fully adhered.

[0059] 3.3 Construction of LPS Damage Model After cell adhesion, the old culture medium was discarded. A blank control group (containing complete culture medium without LPS), a model control group (containing culture medium with LPS), and various drug intervention groups were set up. Except for the blank control group, the other groups were given complete culture medium containing 1 μg / mL LPS and the intervention lasted for 24 h to establish an inflammatory-oxidative damage model of sheep small intestinal epithelial cells.

[0060] 3.4 Gradient Concentration Intervention After successful model establishment, the LPS-containing culture medium was discarded. The drug intervention groups were given complete culture medium containing 5, 10, 20, 40, 80, and 160 μg / mL of condensed tannin from *Caragana korshinskii*, with six replicates per group. The model control group was given complete culture medium without the drug, while the blank control group continued conventional culture. All groups were cultured for another 24 hours.

[0061] 3.5 Indicator Testing (1) Cell viability assay (CCK-8 assay): After culture, add 10 μL of CCK-8 reagent to each well, incubate in the dark for 2 h, and measure the absorbance at 450 nm using a microplate reader. Cell viability (%) = (OD value of experimental wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells) × 100%.

[0062] (2) Detection of inflammatory factors and antioxidant indicators: Cell supernatant was collected from each well of the 96-well plate and the contents of TNF-α and IL-6 (inflammatory factors) and SOD activity, GSH-Px activity and MDA content (oxidative stress indicators) were detected according to the instructions of the ELISA kit.

[0063] (3) Tight junction gene expression detection (qPCR): Cells from each group of 6-well plates were collected, and total RNA was extracted using the TRIzol method and reverse transcribed into cDNA. Using GAPDH as an internal control, the relative mRNA expression levels of Occludin and ZO-1 were detected by qPCR. Primer sequences are as follows:

[0064] Amplification conditions: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles. Relative expression levels were calculated using the 2-ΔΔCt method.

[0065] 3.6 Statistical Analysis One-way ANOVA was performed using SPSS 26.0 software. LSD test was used for comparisons between groups. P<0.05 was considered statistically significant, and P<0.01 was considered extremely significant.

[0066] 3.7 Test Results (1) Cell viability: Compared with the blank control group (100%), the cell viability of the model control group decreased significantly to 58.36%±4.12% (P<0.01). After intervention with condensed tannins from Caragana korshinskii, the cell viability of the 5μg / mL and 10μg / mL groups were 61.05%±3.87% and 63.42%±4.01%, respectively, which were not significantly different from the model control group (P>0.05); the cell viability of the 20μg / mL group increased to 78.95%±3.26% (P<0.05). The survival rate of the 40 μg / mL group reached 89.63% ± 2.85% (P < 0.01), which was the best repair effect; The survival rate in the 80 μg / mL group was 72.18% ± 4.33% (P < 0.05), but it was lower than that in the 40 μg / mL group. The survival rate of the 160 μg / mL group decreased to 45.27% ± 5.10% (P < 0.05), indicating cytotoxicity.

[0067] (2) Inflammatory factors: The levels of TNF-α and IL-6 in the model control group were 4.2 times and 3.8 times higher than those in the blank control group, respectively (P<0.01). After intervention with 40 μg / mL of condensed tannin from *Caragana korshinskii*, TNF-α decreased to 32.5% of the model group (P<0.01), and IL-6 decreased to 28.9% of the model group (P<0.01), showing the most significant anti-inflammatory effect. A significant decrease was also observed at 20 μg / mL (P<0.05), while no significant changes were observed in the 5 and 10 μg / mL groups. The decrease was less pronounced and accompanied by cytotoxicity in the 80 and 160 μg / mL groups.

[0068] (3) Antioxidant indicators: The activities of SOD and GSH-Px in the model control group decreased by 52.3% and 47.6% respectively compared with the blank control group, while the MDA content increased by 3.1 times (P<0.01). The activities of SOD and GSH-Px in the 40μg / mL group recovered to 89.2% and 91.5% of the blank control group, respectively, while the MDA content decreased to 34.6% of the model group (P<0.01), showing the best antioxidant repair effect.

[0069] (4) Tight junction gene expression: The mRNA expression levels of Occludin and ZO-1 in the model control group decreased by 71.4% and 68.9% respectively compared with the blank control group (P<0.01). In the 40 μg / mL group, the expression of Occludin recovered to 82.6% of the blank control group, and the expression of ZO-1 recovered to 79.3% (P<0.01), which significantly repaired the intestinal barrier.

[0070] This embodiment included three independent replicate experiments, with six replicates per group. One-way ANOVA was performed using SPSS 26.0, with LSD test. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. The RSD for repeatability was < 5%. Results showed that the cell viability in the 40 μg / mL group was 89.63 ± 2.85%, TNF-α decreased to 32.5% of the model group, IL-6 decreased to 28.9%, SOD recovered to 89.2% of the control group, and Occludin expression increased to 82.6%. The overall effect was optimal with no toxicity.

[0071] 3.8 Conclusion Based on the above four-dimensional indicators, the repair effect of condensed tannins from *Caragana korshinskii* on LPS-induced damage to sheep small intestinal epithelial cells exhibits a strictly concentration-dependent effect: low concentrations (5, 10 μg / mL) are ineffective; medium and high concentrations (20, 40 μg / mL) are effective, with 40 μg / mL being the optimal in vitro effective and safe concentration, combining the best repair effect with no cytotoxicity; high concentrations (80, 160 μg / mL) show cytotoxicity. This concentration parameter is being disclosed quantitatively for the first time.

[0072] Example 4: In vivo feeding verification test of the effect of condensed tannin preparations of Caragana korshinskii on the growth performance of Dumont lambs. 4.1 Test Materials Caragana condensed tannin preparation: prepared by combining 96.2% pure Caragana condensed tannin obtained from Example 1 with corn starch carrier, with an active tannin content of 8%.

[0073] Experimental animals: Healthy, disease-free Dorper lambs (F1 generation of Dorper × Mongolian sheep cross), aged 1-2 months, with similar weights, were purchased from large-scale meat sheep breeding bases in Inner Mongolia.

[0074] Basic diet: Complete lamb compound feed, formulated according to the NRC (2007) nutritional requirements for sheep, without any antibiotics or tannin additives.

[0075] 4.2 Experimental Design A single-factor completely randomized design was used to select 36 Dumont lambs (half male and half female) and randomly divide them into 2 groups with 3 replicates in each group and 6 lambs in each replicate.

[0076] Control group: fed with a basal diet.

[0077] Experimental group: 0.1% (mass fraction, equivalent to about 40mg / kg body weight per animal per day, converted from the optimal concentration in previous cell experiments) of condensed tannins of *Caragana korshinskii* were added to the basal diet.

[0078] Experimental period: 7 days for the preliminary trial and 60 days for the formal trial. During the trial, the animals had free access to food and water, and the rearing environment, lighting, ventilation, and disease prevention management remained consistent.

[0079] 4.3 Measurement Indicators and Methods Weight: The initial weight is determined by weighing yourself on an empty stomach on the morning of the first day of the trial period, and the final weight is determined by weighing yourself on an empty stomach on the 60th day.

[0080] Feed intake: Record the amount of feed given and the amount of feed left over for each group daily, and calculate the average daily feed intake (ADFI) for each sheep.

[0081] Growth performance calculation: Average daily weight gain (ADG, g / d) = (final weight - initial weight) / 60; Feed conversion ratio (F / G) = ADFI / ADG.

[0082] 4.4 Data Statistics Independent samples t-tests were performed using SPSS 26.0 software. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered highly statistically significant. Data are expressed as mean ± standard deviation.

[0083] 4.5 Test Results Table 2. Effects of Caragana condensed tannins on growth performance of Dumont lambs.

[0084] Table 2 shows that there was no significant difference in initial weight between the two groups of lambs (P>0.05), indicating a balanced grouping. The final weight and average daily weight gain of the experimental group were significantly higher than those of the control group (P<0.05), with an ADG increase of 27.4%. There was no significant difference in average daily feed intake between the two groups (P>0.05). The feed conversion ratio of the experimental group was significantly lower than that of the control group (P<0.05), with a feed conversion efficiency increase of 20.2%.

[0085] 4.6 Discussion and Conclusion In vivo feeding trials showed that, without significant changes in feed intake, dietary supplementation with condensed tannins from *Caragana korshinskii* significantly improved daily weight gain, reduced feed conversion ratio, and enhanced growth performance in Dorset lambs. Combined with the cellular mechanism study in Example 3, condensed tannins from *Caragana korshinskii* reduced subclinical intestinal damage induced by endotoxins such as LPS through anti-inflammatory, antioxidant, and intestinal epithelial barrier repair effects, thereby improving nutrient digestibility and absorption efficiency and ultimately enhancing growth efficiency without increasing feed intake. These results validate the effectiveness of the 40 μg / mL (in vitro) in vivo supplementation level selected in this invention.

[0086] The condensed tannin powder preparation of *Caragana korshinskii* prepared by this invention can be directly and uniformly mixed into the basic diet of lambs in large-scale sheep farming at a mass fraction of 0.1%. Long-term feeding can alleviate intestinal epithelial inflammatory damage caused by stress and pathogenic bacterial toxins, improve nutrient absorption efficiency, increase daily weight gain, and reduce feed conversion ratio. At the same time, the in vitro screening method of this invention can be used to conduct efficacy tests on preparations made from different batches of *Caragana korshinskii* raw materials, and qualified products can be screened before being put into breeding applications, so as to achieve controllable product quality.

[0087] In summary, this invention provides a condensed tannin preparation of *Caragana korshinskii* for repairing inflammatory damage to sheep small intestinal epithelial cells and a method for screening its effective concentration. A standardized in vitro concentration screening system has been established, and its effect on improving sheep growth performance has been confirmed through live feeding experiments, demonstrating significant prospects for industrial application.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A condensed tannin preparation of *Caragana korshinskii* for repairing inflammatory damage to sheep small intestinal epithelial cells, characterized in that, The active ingredient is condensed tannin of Caragana korshinskii with a purity of ≥95%; the condensed tannin of Caragana korshinskii is obtained by extracting the stems and leaves of Caragana korshinskii with 70% acetone and eluting with D101 macroporous resin gradient purification; the safe and effective concentration of the preparation acting on the small intestinal epithelial cells of immortalized sheep is 40 μg / mL; the addition ratio in the diet of Dumont lambs is 0.1%.

2. The formulation according to claim 1, characterized in that, The formulation is a powder, the excipient is corn starch / wheat bran carrier, and the effective tannin content is 5%~10% by mass.

3. The formulation according to claim 1, characterized in that, The purification process of condensed tannins from Caragana korshinskii involves a material-to-liquid ratio of 1:15, extraction at 50°C for 2 hours, followed by washing with D101 resin to remove impurities and elution with 70% acetone.

4. A method for screening the effective concentration of the condensed tannin preparation of *Caragana korshinskii* as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Immortalized sheep small intestinal epithelial cells were injected at a concentration of 1×10⁻⁶. 5 Inoculate at a density of 100 cells / mL into culture plates and incubate for 12 hours until fully adherent; (2) Except for the blank control group, Escherichia coli LPS with a final concentration of 1 μg / mL was added for 24 h to construct an inflammatory-oxidative complex injury model; (3) Discard the LPS medium and add a complete medium containing a gradient concentration of Caragana condensed tannin for 24 h. The gradient concentrations are 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL and 160 μg / mL, respectively. (4) Cell viability was detected by CCK-8 assay, inflammatory factors TNF-α and IL-6 and antioxidant indicators SOD, GSH-Px and MDA were detected by ELISA, and the expression of tight junction genes Occludin and ZO-1 was detected by qPCR. (5) Based on the detection results of step (4), the concentrations that significantly improve cell survival rate, reduce inflammatory factors, improve antioxidant capacity, upregulate tight junction gene expression and have no cytotoxicity are selected and determined as the optimal in vitro effective and safe concentrations.

5. The method according to claim 4, characterized in that, The LPS mentioned in step (2) is a lipopolysaccharide derived from Escherichia coli O55:B5.

6. The method according to claim 4, characterized in that, In step (4), the inflammatory factors TNF-α and IL-6 were quantitatively detected using a double-antibody sandwich ELISA method; the antioxidant indicators SOD activity, GSH-Px activity and MDA content were detected using an ELISA kit; and the relative expression levels of the tight junction genes Occludin and ZO-1 were detected using real-time quantitative qPCR.

7. The method according to claim 4, characterized in that, The optimal in vitro effective safe concentration mentioned in step (5) is 40 μg / mL.

8. The method according to claim 4, characterized in that, The immortalized sheep small intestinal epithelial cells were constructed through the following steps: Select jejunal tissue from healthy newborn lambs, peel off the serosa and muscle layers, rinse with PBS, and cut into 1mm pieces. 3 The fragments were digested with 0.25% trypsin-EDTA for 20-30 minutes, then filtered and centrifuged to obtain primary cells; The human telomerase reverse transcriptase hTERT gene was introduced using lentiviral transfection. After transfection, the cells were selected with 400 μg / mL G418 for 10-14 days, and single clones were selected for amplification and culture. After more than 30 passages, immortalized sheep small intestinal epithelial cell lines were obtained.

9. The method according to claim 4, characterized in that, The extraction and purification process of the condensed tannins from *Caragana korshinskii* includes: using a 70% acetone aqueous solution at a material-to-liquid ratio of 1:15 (g:mL), extracting at 50℃ for 2 hours, and collecting the crude extract by centrifugation; using D101 macroporous adsorption resin, loading the sample at 2.0mL / min, eluting with ultrapure water at 3.0mL / min to remove impurities, and then eluting with a 70% acetone aqueous solution at a gradient rate of 1.5mL / min; collecting the eluent, concentrating under reduced pressure, and vacuum drying to obtain condensed tannins from *Caragana korshinskii* with a purity ≥95%.

10. The method according to claim 4, characterized in that, The specific operation of the CCK-8 method for detecting cell viability in step (4) is as follows: add 10 μL of CCK-8 reagent to each well, incubate in the dark for 2 h, detect the absorbance value at a wavelength of 450 nm, and calculate the cell viability.

Images