Application of prunella vulgaris phenolic acid in preparation of product for preventing, treating and / or relieving liver injury complicated with uveitis

Abstract

The invention relates to an application of prunella vulgaris phenolic acid in preparation of a product for preventing, treating and / or relieving liver injury complicated with uveitis. Researches show that prunella vulgaris phenolic acid improves the symptoms such as vasodilatation, iris congestion and pupil tightening, obviously reduces the eye score, can effectively improve D-GalN / LPS induced retinopathy, effectively reduces the content of ALT and AST in serum and improves D-GalN / LPS induced liver injury, and it is indicated that prunella vulgaris phenolic acid can relieve and treat liver injury combined uveitis. The application provides a new application for prunella vulgaris phenolic acid, and provides a new product for preventing, treating and / or relieving D-GalN / LPS induced liver injury combined uveitis at the same time.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of prunella vulgaris phenolic acid in the preparation of products for preventing, treating and / or alleviating liver damage combined with uveitis. Background Art

[0002] Liver injury combined with uveitis refers to liver and eye damage induced by the combined use of D-GalN (D-galactosamine) and LPS (lipopolysaccharide). The specific pathological symptoms are similar. For example, in the liver, it can cause liver cell necrosis and inflammatory response, and in the eye, it can cause corneal opacity, retinal lesions and other injuries, thereby affecting the normal function of related organs.

[0003] Current prevention and treatment methods for liver injury combined with uveitis have numerous limitations. For example, while traditional anti-inflammatory drugs, such as glucocorticoids (e.g., dexamethasone), are somewhat effective in suppressing inflammatory responses, long-term or excessive use can be associated with numerous side effects. In the liver, these drugs can lead to abnormal liver function, such as elevated levels of alanine aminotransferase and aspartate aminotransferase, and may also cause pathologies such as fatty liver disease and liver fibrosis. In the eyes, these drugs can increase intraocular pressure, cataracts, glaucoma, and other eye diseases, impairing vision. Traditional prevention and treatment methods are associated with significant side effects, suboptimal efficacy, and long treatment cycles, as well as a lack of specific, effective preventive measures. Therefore, there is a need for a new medication that can effectively prevent, treat, and / or alleviate liver injury combined with uveitis. Summary of the Invention

[0004] Based on this, the present application provides a new use of prunella vulgaris phenolic acid, which relates to the use of prunella vulgaris phenolic acid in the preparation of products for preventing, treating and / or alleviating liver damage combined with uveitis.

[0005] The specific technical solutions are as follows:

[0006] In a first aspect of the present application, there is provided a use of prunella vulgaris phenolic acid in the preparation of a product for preventing, treating and / or alleviating liver damage combined with uveitis.

[0007] This application discovered through research that prunella vulgaris phenolic acid improves symptoms such as vasodilation, iris congestion, and pupil constriction, significantly reduces ocular scores, and can effectively improve D-GalN / LPS-induced retinopathy. It also effectively reduces serum ALT and AST levels and improves D-GalN / LPS-induced liver damage, indicating that prunella vulgaris phenolic acid can alleviate and treat liver damage combined with uveitis. This application provides a new use for prunella vulgaris phenolic acid and a new product for preventing, treating, and / or alleviating D-GalN / LPS-induced liver damage combined with uveitis.

[0008] Studies have found that phenolic acid can be used to prepare products for preventing, treating and / or alleviating liver damage combined with uveitis. It has obvious efficacy, broad application prospects, and great value for promotion and application.

[0009] In some embodiments, the prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of IL-6 inflammatory factor.

[0010] In some embodiments, the prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of IL-1β inflammatory factor.

[0011] In some embodiments, the prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of TNFα inflammatory factor.

[0012] In some embodiments, the dosage forms of the product for preventing, treating and / or alleviating liver damage combined with uveitis include solutions, powders, tablets, granules, capsules, emulsions and suspensions.

[0013] In some embodiments, the dosage form of the product for preventing, treating and / or alleviating liver injury combined with uveitis is a solution, mainly composed of prunella vulgaris acid and a solvent; the solvent includes at least one of dimethyl sulfoxide, polyethylene glycol 300, Tween-80 and normal saline.

[0014] In some embodiments, the phenolic acid is derived from at least one of Prunella vulgaris ears, stems, leaves, and seeds.

[0015] The second aspect of the present application further provides a drug for preventing, treating and / or alleviating liver damage combined with uveitis, wherein the active ingredient of the drug for preventing, treating and / or alleviating liver damage combined with uveitis includes prunella vulgaris phenolic acid.

[0016] In some embodiments, the drug for preventing, treating and / or alleviating liver damage combined with uveitis is in the form of an oral or injectable dosage form.

[0017] In some embodiments, the oral dosage forms include solutions, powders, tablets, granules, capsules, emulsions and suspensions;

[0018] And or, the injection dosage form includes solution and suspension. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The HPLC chromatograms of the Prunella vulgaris fruit spike phenolic acid test sample, the Prunella vulgaris stem and leaf phenolic acid test sample, the Prunella vulgaris seed phenolic acid test sample, and the mixed reference substance in Example 1 are shown;

[0020] Figure 2These are preganglionic photographs of the rat eyes of each group in Example 1;

[0021] Figure 3 This is a graph showing the eye scoring results of rats in each group in Example 1;

[0022] Figure 4 This is the liver index result diagram of each group of rats in Example 1;

[0023] Figure 5 Figure 1 shows the liver function results of rats in each group in Example 1

[0024] Figure 6 The following are the results of HE staining of the eyes of rats in each group in Example 1;

[0025] Figure 7 The results of HE staining of the liver of rats in each group in Example 1 are shown;

[0026] Figure 8 ALT and AST levels in serum in Example 1;

[0027] Figure 9 Figure 1 is the WB results of S100A9 in the liver and retina of each group of rats in Example 1;

[0028] Figure 10 The figure shows the results of the acylcarnitine content in the liver and retina of rats in each group in Example 1;

[0029] Figure 11 The figure shows the results of the acylcarnitine content in the liver and retina of rats in each group in Example 1;

[0030] Figure 12 This is a graph showing the acylcarnitine content in the liver and retina of rats in each group in Example 1. DETAILED DESCRIPTION

[0031] The following, with reference to specific examples, further details the use of the prunella vulgaris phenolic acid described herein in preparing products for preventing, treating, and / or alleviating liver damage complicated by uveitis. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to facilitate a more thorough and comprehensive understanding of the disclosure herein.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0033] The optional scope of the terms "and / or", "or / and", and "and / or" used in this document includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0034] In this application, terms such as "first aspect" and "second aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, terms such as "first" and "second" serve only as non-exhaustive enumeration and description and should not constitute a closed-ended limitation on quantity.

[0035] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0036] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the weights described in the examples of this application may be mass units known in the chemical industry, such as μg, mg, g, and kg.

[0037] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two numerical endpoints. In this document, this is equivalent to directly listing each integer. For example, "t is an integer selected from 1-10" means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges may be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0038] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control.

[0039] D-Galactosamine is an amino sugar that, under normal physiological conditions, undergoes a specific metabolic pathway. However, when administered at a certain dose, D-GalN can interfere with certain metabolic processes within liver cells, particularly those closely related to protein and nucleic acid synthesis and metabolism. For example, it can deplete uridine diphosphate-galactose (UDP-Gal) within hepatocytes, thereby affecting the synthesis of various galactose-containing glycoproteins and glycolipids, leading to disruption of the normal structure and function of hepatocytes.

[0040] Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacterial cell walls, is highly immunogenic. Once inside the body, LPS is recognized by corresponding receptors on the surface of immune cells, activating a series of immune response signaling pathways. LPS can induce an inflammatory response in corneal epithelial and stromal cells, prompting them to release inflammatory factors such as TNF-α and IL-1β. These inflammatory factors increase the permeability of corneal epithelial cells, weakening their barrier function and making them susceptible to invasion by external pathogens.

[0041] In the liver, D-GalN / LPS-induced liver enlargement and hardening may occur. D-GalN interferes with hepatocyte metabolism, impairing its synthetic function. Furthermore, LPS-activated inflammatory factors further burden hepatocytes, disrupting their normal metabolic processes. For example, they inhibit mitochondrial respiration, leading to disrupted energy metabolism. Histopathological examinations reveal extensive hepatocyte necrosis, inflammatory cell infiltration, and disrupted lobular architecture. Significant abnormalities in liver function markers, such as elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin, may also be observed, reflecting hepatocyte damage and impaired liver function. Corneal opacity, edema, and corneal epithelial cell damage and detachment may be observed. Retinal edema and hemorrhage, a decrease and degeneration of retinal ganglion cells, and pigment epithelial cell dysfunction may occur.

[0042] Prunella vulgaris acid can effectively improve D-GalN / LPS-induced liver injury combined with uveitis, achieving "co-repair" of the liver and retina. The "co-material basis" is acylcarnitine, and the "co-action mechanism" is that PVPs can inhibit the expression of S100A9, reduce the accumulation of acylcarnitine, and inhibit the inflammatory response of the liver and retina.

[0043] The present application provides the use of prunella vulgaris phenolic acid in the preparation of a product for preventing, treating and / or alleviating liver damage combined with uveitis.

[0044] In some embodiments, the method for preparing the above-mentioned prunella vulgaris phenolic acid includes the following steps S10 to S30.

[0045] Step S10: heating the Prunella vulgaris sample with ethanol having a volume fraction of 20% to 80% and refluxing for 0.5 h to 1.5 h, performing solid-liquid separation, and preparing a filtrate containing Prunella vulgaris phenolic acid.

[0046] In some embodiments, the solid-to-liquid ratio of the above-mentioned Prunella Vulgaris sample to ethanol with a volume fraction of 30% to 80% is 1: (5 to 20).

[0047] In a specific example, the solid-to-liquid ratio of the above-mentioned Prunella Vulgaris sample to ethanol with a volume fraction of 30% to 80% is 1:10.

[0048] In some embodiments, the above-mentioned self-phenolic acid is derived from at least one of self-phenol panicles, self-phenol stems and leaves, and self-phenol seeds.

[0049] Prunella vulgaris fruit spike phenolic acid, Prunella vulgaris stem and leaf phenolic acid, and Prunella vulgaris seed phenolic acid can effectively improve D-GalN / LPS-induced liver injury combined with uveitis, achieving "co-repair" of the liver and eyes. The "co-material basis" is acylcarnitine, and the "co-action mechanism" is that PVPs can inhibit the expression of S100A9, reduce the accumulation of acylcarnitine, and inhibit the inflammatory response of the liver and retina. Prunella vulgaris seed phenolic acid has a better therapeutic effect.

[0050] Step S20: Concentrating the filtrate containing the prunella vulgaris phenolic acid to prepare a concentrated solution containing the prunella vulgaris phenolic acid.

[0051] Step S30: enriching the concentrated solution containing prunella vulgaris phenolic acid with a macroporous adsorption resin, eluting with ethanol with a volume fraction of 20% to 70%, collecting the eluate, and sequentially concentrating and drying to obtain prunella vulgaris phenolic acid.

[0052] In some embodiments, the model of the macroporous adsorption resin includes at least one of AB-8, NKA-9, S-8, DM-130, HPD-100, and D-101.

[0053] In a specific example, the model of the macroporous adsorption resin is AB-8.

[0054] In some embodiments, the elution flow rate is 3 mL / min to 8 mL / min.

[0055] In a specific example, the elution flow rate is 5 mL / min.

[0056] In some embodiments, after the perforated adsorption resin enrichment step, the method further includes eluting the product enriched with the macroporous adsorption resin with water and discarding the eluent before the elution step with 20% to 70% ethanol by volume.

[0057] In one specific example, the above-mentioned prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of IL-6 inflammatory factor.

[0058] In one specific example, the above-mentioned prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of IL-1β inflammatory factor.

[0059] In one specific example, the above-mentioned prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of TNFα inflammatory factor.

[0060] In some embodiments, the dosage forms of the above-mentioned product for preventing, treating and / or alleviating liver damage combined with uveitis include solutions, powders, tablets, granules, capsules, emulsions and suspensions.

[0061] In a specific example, the dosage form of the product for preventing, treating and / or alleviating liver damage combined with uveitis is a solution, which is mainly composed of prunella vulgaris acid and a solvent.

[0062] Specifically, the solvent includes at least one of dimethyl sulfoxide, polyethylene glycol 300, Tween-80 and physiological saline, but is not limited thereto.

[0063] In one embodiment, the product further comprises one or more of a binder, a lubricant, a glidant, a diluent and a disintegrant.

[0064] In one embodiment, the binder includes one or more of povidone K30, copovidone, sodium carboxymethylcellulose, hydroxypropyl cellulose and hypromellose.

[0065] In one embodiment, the lubricant includes one or more of magnesium stearate, talc and polyethylene glycol.

[0066] In one embodiment, the glidant includes one or more of silicon dioxide, micro-powdered silica gel, calcium hydrogen phosphate, tricalcium phosphate and milk mineral salt.

[0067] In one embodiment, the diluent comprises one or more of microcrystalline cellulose, isomalt, starch, dextrin, lactose, pregelatinized starch, mannitol, sorbitol and erythritol.

[0068] In one embodiment, the disintegrant comprises one or more of crospovidone, sodium starch glycolate, croscarboxymethyl cellulose sodium, low-substituted hydroxypropyl cellulose and dry starch.

[0069] In some embodiments, the method for preparing the product for preventing, treating, and / or alleviating liver injury combined with uveitis comprises the following steps: mixing prunella vulgaris phenolic acid with a solvent to prepare the product for preventing, treating, and / or alleviating liver injury combined with uveitis. It will be appreciated that the preparation method may be adjusted as needed depending on the dosage form or formulation.

[0070] In some embodiments, the phenolic acid is derived from at least one of the fruit clusters, stems, leaves, and seeds of Prunella vulgaris. This application also provides an animal model for validating the aforementioned product for preventing, treating, and / or alleviating liver damage combined with uveitis. The animal model is a liver and eye damage model induced by intraperitoneal injection of 100 mg / kg to 200 mg / kg of D-galactosamine and 1 mg / kg of lipopolysaccharide.

[0071] In a specific example, the animal model is a liver and eye damage model induced by intraperitoneal injection of 150 mg / kg D-galactosamine and 1 mg / kg lipopolysaccharide.

[0072] This application discovered through research that prunella vulgaris phenolic acid improves symptoms such as vasodilation, iris congestion, and pupil constriction, significantly reduces ocular scores, and can effectively improve D-GalN / LPS-induced retinopathy. It also effectively reduces serum ALT and AST levels and improves D-GalN / LPS-induced liver damage, indicating that prunella vulgaris phenolic acid can alleviate and treat liver damage combined with uveitis. This application provides a new use for prunella vulgaris phenolic acid and a new product for preventing, treating, and / or alleviating D-GalN / LPS-induced liver damage combined with uveitis.

[0073] Studies have found that phenolic acid can be used to prepare products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye diseases. It has obvious efficacy, broad application prospects, and great promotion and application value.

[0074] One embodiment of the present application further provides a drug for preventing, treating and / or alleviating liver damage combined with uveitis. The active ingredient of the drug for preventing, treating and / or alleviating liver damage combined with uveitis includes prunella vulgaris acid.

[0075] In some embodiments, the dosage form of the drug for preventing, treating and / or alleviating liver damage combined with uveitis is an oral or injectable dosage form.

[0076] In some embodiments, the oral dosage forms include solutions, powders, tablets, granules, capsules, emulsions and suspensions.

[0077] In some embodiments, the above-mentioned injection dosage forms include solutions and suspensions.

[0078] It is understood that the above-mentioned drugs for preventing, treating and / or alleviating liver damage combined with uveitis can be administered by subcutaneous injection, intracerebral administration, intravenous injection, oral administration, etc.

[0079] The above-mentioned drugs for preventing, treating and / or alleviating liver damage combined with uveitis can reduce liver biochemical indicators, serum, liver and retinal inflammatory factor levels in model rats, reduce ocular inflammatory infiltration, and alleviate liver damage combined with uveitis.

[0080] The following are specific embodiments

[0081] Unless otherwise specified, the reagents and instruments used in the examples are all conventionally selected in the art. Experimental methods without specific conditions specified in the examples were performed according to conventional conditions, such as those described in literature or books or methods recommended by manufacturers.

[0082] Example 1

[0083] 1. Preparation of experimental materials

[0084] 1.1 SD rats and housing conditions:

[0085] SPF-grade SD male rats were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. with license number GYS-201905230001.

[0086] The experimental animals were housed in the Experimental Animal Center of Hunan University of Chinese Medicine with approval from the Ethics Committee of Hunan University of Chinese Medicine. The experimental unit use permit number is SYXK (Xiang) 2019-0009.

[0087] 1.2 Rearing conditions: temperature 22-24℃, humidity 60-65%, day and night alternation, free access to food and water.

[0088] 1.3 Experimental Reagents:

[0089] LPS (lipopolysaccharide), D-Gal (D-galactose), anesthetics: use appropriate anesthetics such as chloral hydrate, 4% paraformaldehyde fixative, eyeball fixative, hematoxylin staining solution, eosin staining solution, TNF-α ELISA Kit, IL-6 ELISA Kit, IL-1β ELISA Kit, sodium chloride injection, Prunella vulgaris fruit spikes, Prunella vulgaris stems and leaves, and Prunella vulgaris seeds.

[0090] 2. Experimental methods

[0091] 2.1 Preparation of LPS / D-Gal mixed solution:

[0092] Calculate the amount of LPS and D-Gal according to the required concentrations, weigh the required amount of LPS and D-Gal respectively, add them to physiological saline, mix well, dissolve by ultrasonication, and store in a 4°C refrigerator for later use.

[0093] 2.2 Preparation of Prunella Vulgaris Spike Phenolic Acid, Stem Leaf Phenolic Acid, and Seed Phenolic Acid:

[0094] The panicles, stems, leaves and seeds of Prunella vulgaris were crushed and sieved, and extracted three times with 10 volumes of 70% ethanol by heating and reflux for 1 hour. The extracts were filtered, combined and concentrated until there was no alcohol taste, and then transferred to an AB-8 macroporous resin chromatography column (solid-liquid ratio of 10:1). Five column volumes of pure water and 50% ethanol were added in sequence for elution (elution flow rate of 5 mL / min). The 50% ethanol fraction was collected, concentrated and freeze-dried to obtain Prunella vulgaris panicle phenolic acids, stem and leaf phenolic acids and seed phenolic acids.

[0095] 2.2.1 UHPLC-Q-TOF-MS / MS component characterization

[0096] 2.2.1.1 Preparation of test solution

[0097] 50 mg of sample powder was accurately weighed using an analytical balance and placed in a 2-mL EP tube. Two homogenizing grinding beads were added, and 500 μL of extraction solution (methanol:acetonitrile:water (2:2:1, v / v / v)) containing an isotope-labeled internal standard was added. The tube was vortexed for 30 s and homogenized in a homogenizer (35 Hz, 240 s). The tube was then transferred to an ice-water bath and sonicated for 5 min. The homogenization and sonication steps were repeated three times. The tube was placed in a −40°C refrigerator and allowed to stand for 30 min. The tube was centrifuged at 12,000 rpm (13,800 × g, radius 8.6 cm) for 15 min at 4°C, and the supernatant was removed. The supernatant was placed in a −40°C refrigerator for 10 min and centrifuged at 12,000 rpm (13,800 × g, radius 8.6 cm) for 15 min at 4°C. The supernatant was filtered through a 0.22 μm microporous filter into a vial for analysis.

[0098] 2.2.1.2 Detection methods

[0099] The target compounds were separated chromatographically on a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph. Phase A consisted of aqueous phase containing 0.01% acetic acid, and phase B consisted of isopropanol:acetonitrile (1:1, v / v). The gradient elution was: 1% B (0–1 min), 1%–99% B (1–8 min), 99% B (8–9 min), 99%–1% B (9–9.1 min), and 1% B (9.1–12 min). The sample tray temperature was 4°C, the injection volume was 2 μL, and the flow rate was 0.3 mL / min. -1 .

[0100] An Orbitrap Exploris 120 mass spectrometer was capable of acquiring both primary and secondary mass spectrometric data under the control of Xcalibur software (version 4.4, Thermo). Detailed parameters were as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320°C, Full MS resolution: 60,000, MS / MS resolution: 15,000, Collision energy: SNCE 20 / 30 / 40, Spray voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0101] 2.2.2 HPLC quantitative analysis

[0102] 2.2.2.1 Preparation of reference solution

[0103] Weigh approximately 2.10 mg of caffeic acid, 5.40 mg of isorosmarinic acid, and 5.30 mg of rosmarinic acid accurately and prepare stock solutions using 50% methanol. Draw an appropriate amount of each of the three reference solutions and dilute with 50% methanol to create a mixed reference solution.

[0104] 2.2.2.2 Preparation of test solution

[0105] Take about 10.0 mg, 10.5 mg, and 10.8 mg of phenolic acid from the spike of Prunella vulgaris, stem and leaf of Prunella vulgaris, and seed of Prunella vulgaris, accurately weigh them, and use 50% methanol as the solvent to prepare test solutions of corresponding concentrations.

[0106] 2.2.2.3 HPLC detection method

[0107] Chromatographic conditions: Column: Agilent SB-C18 (4.6 mm × 250 mm, 5 μm); Mobile phase: methanol (A)-0.1% formic acid solution (B); Gradient elution: 5% - 35% A (0-15 min), 35% A (15-40 min), 35% - 55% A (40-45 min); Flow rate: 1.0 mL min -1 ; Wavelength 330 nm; Column temperature 30℃; Injection volume 10 μL.

[0108] 2.2.3 Content determination results

[0109] 2.2.3.1 Methodological investigation

[0110] (1) Linear relationship investigation: Caffeic acid in the range of 0.00042-0.0021 mg·mL -1 There is a good linear relationship within the range, y = 49152x - 1.248 (R² = 0.9999).

[0111] Isorosmarinic acid glycosides were 0.025327385-0.405238154 mg·mL -1 There is a good linear relationship within the range, y = 42994x + 52.509 (R² = 0.9999).

[0112] Rosmarinic acid at 0.053-0.53 mg·mL -1 The linear relationship is good within the range of y = 55219x - 159.13 (R² = 0.9996).

[0113] The contents of caffeic acid, isorosmarinic acid glycosides and rosmarinic acid in Prunella vulgaris fruit spike phenolic acid, Prunella vulgaris stem leaf phenolic acid and Prunella vulgaris seed phenolic acid were determined (the results of liquid phase are shown in Table 1 and Figure 1 , Figure 1 The HPLC chromatograms of the Prunella vulgaris spike phenolic acid sample (A), Prunella vulgaris stem and leaf phenolic acid sample (B), Prunella vulgaris seed phenolic acid sample (C), and mixed reference sample (D) are shown. 1 represents caffeic acid, 2 represents isorosmarinic acid glycosides, and 3 represents rosmarinic acid. The contents of caffeic acid, isorosmarinic acid glycosides, and rosmarinic acid in the Prunella vulgaris spike phenolic acid were 4.25 mg·g, respectively. -1 、41.94 mg·g -1 、159.05 mg·g -1 The contents of caffeic acid, isorosmarinic acid glycosides and rosmarinic acid in the stems and leaves of Prunella vulgaris were 0.97 mg·g -1 、3.65 mg·g -1、137.55 mg·g -1 The contents of caffeic acid, isorosmarinic acid glycosides and rosmarinic acid in Prunella vulgaris seeds were 0.33 mg·g -1 、258.98 mg·g -1 、234.57 mg·g -1 .

[0114] Table 1 Contents of caffeic acid, isorosmarinic acid glycosides and rosmarinic acid in the panicles, stems, leaves and seeds of Prunella vulgaris

[0115]

[0116] 2.3 Animal Experimental Procedures

[0117] Thirty-six 6-week-old SD rats were randomly divided into 6 groups after 7 days of adaptive feeding (weight was controlled at about 230 g), with 6 rats in each group.

[0118] Grouping and treatment: Normal group, no treatment; model group, intraperitoneal injection of D-GalN / LPS mixed solution on the 14th day; dexamethasone group, intraperitoneal injection of dexamethasone solution (10 mg / kg) on the 14th day, 2 hours after intraperitoneal injection of D-GalN / LPS mixed solution; Prunella vulgaris spike phenolic acid group, intraperitoneal administration of 200 mg / kg Prunella vulgaris spike phenolic acid every day for 7 consecutive days; Prunella vulgaris stem and leaf phenolic acid group, intraperitoneal administration of 200 mg / kg Prunella vulgaris stem and leaf phenolic acid every day for 7 consecutive days; Prunella vulgaris seed phenolic acid group, intraperitoneal administration of 200 mg / kg Prunella vulgaris seed phenolic acid every day for 7 consecutive days, and intraperitoneal injection of D-GalN / LPS mixed solution 2 hours after intraperitoneal injection on the 14th day.

[0119] Preparation of related solutions:

[0120] (1) Preparation of LPS / D-GalN mixed solution: Calculate the amount of LPS and D-GalN according to the required concentrations. Weigh the required amount of LPS and D-GalN, prepare LPS at 0.1 mg / ml and D-GalN at 15 mg / ml, add them to physiological saline, mix well, dissolve by ultrasonication, and store in a refrigerator at 4°C for later use.

[0121] (2) Prunella vulgaris fruit acid, Prunella vulgaris stem and leaf phenolic acid, and Prunella vulgaris seed phenolic acid: The dosage is 200 mg / kg, and the dosing volume for rats is 5 mL / kg to 10 mL / kg, and normal saline is added for ultrasonic dissolution.

[0122] (3) Dexamethasone sodium phosphate injection: Add normal saline to prepare 0.1 mg / mL, the dosage is 1 mg / kg.

[0123] 2.4 Sample Collection

[0124] 18 hours after intraperitoneal injection, rats in each group were observed and photographed using a portable slit lamp microscope. Rats were anesthetized with intraperitoneal injection of chloral hydrate. Blood was collected from the abdominal aorta, and the liver was removed. After weighing, a portion was fixed in 4% paraformaldehyde, and the remaining tissue was quickly stored in a -80°C freezer. The right eyeball was removed and fixed in eye fixative. The left retina was dissected under a stereomicroscope and stored in a -80°C freezer. The collected blood was allowed to rest for approximately 30 minutes, centrifuged at 3400 rpm for 10 minutes, and the supernatant was removed with a pipette to obtain the serum.

[0125] 2.5 Rat eye scoring

[0126] The rats' eyes were observed under a slit lamp microscope and scored for iris dilation and congestion, pupil constriction, fibrous exudation or adhesion in the anterior chamber of the pupil, and hypopyon. The severity of ocular symptoms was analyzed based on the combined score. The scoring criteria are shown in Table 2.

[0127] Table 2 Ocular symptom scoring table

[0128]

[0129] 2.6 Paraffin embedding and sectioning of rat liver and eye tissues

[0130] ① Tissue dehydration: After removing the rat liver tissue and eyeball from the fixative, rinse with tap water for 15 minutes and then place them in 75%, 85%, 95% and 100% ethanol for 5 minutes each.

[0131] ② Tissue transparency: Soak the tissue in a xylene-ethanol (1:1) solution for 15 minutes, then soak it in pure xylene for 15 minutes, and finally replace it with new xylene and soak it for 15 minutes.

[0132] ③ Paraffin-embedded tissue: Soak in wax in a 65°C constant temperature drying oven for 2 hours, place the liver tissue and eyeball in an embedding basket and embed to make wax blocks.

[0133] ④ Serially slice the wax block using a tissue slicer and observe the slices under a microscope. Bake in a 65°C constant temperature drying oven for 30 minutes, cool to room temperature, transfer to a slice box, and store in a 4°C refrigerator.

[0134] 2.7 Rat liver index determination

[0135] After blood was collected from the abdominal aorta of the rats, the liver was removed, cleaned in physiological saline, and weighed. The liver index of each rat was calculated according to the following formula.

[0136]

[0137] 2.8 Determination of rat liver function indicators

[0138] 300 μL of serum from each group of rats was collected and placed in EP tubes. The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were tested using an automatic biochemical analyzer.

[0139] 2.9 HE staining of rat liver and eye tissues

[0140] Place the cut paraffin sections in an oven and bake at 65°C for 1 hour until the paraffin melts, then soak the sections in xylene I for 20 minutes; then transfer the sections to xylene II and soak for 20 minutes; after the wax on the sections dissolves, transfer the sections to anhydrous ethanol I and soak for 5 minutes, then transfer to anhydrous ethanol II and soak for 5 minutes, then scrub with anhydrous ethanol for 20 seconds, transfer to a basin of water and rinse off the alcohol on the samples with running water.

[0141] Place the dewaxed sections in a hematoxylin staining jar for 3-5 minutes. Remove the sections and rinse with water from the staining cup to remove excess hematoxylin. Place the sections in the differentiation solution in the staining jar for 3-5 seconds, rinse quickly with water, and then place the sections in the bluing solution in the staining jar for 3-5 seconds, rinse quickly with water. Then, place the sections in the staining jar in the following order: 85% ethanol, 95% ethanol, eosin solution, anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, n-butanol, xylene I, and xylene II, soak for 3-5 minutes each. Remove the sections and quickly air dry them in an air vent before mounting them with neutral gum.

[0142] 2.10 ELISA determination of TNF-α, IL-6, and IL-1β in rat serum

[0143] (1) Index determination

[0144] a) Remove the reagent kit from the refrigerator and allow it to cool to room temperature. Prepare standard solutions of varying concentrations using a gradient: IL-6 standard at 800 pg / mL, 400 pg / mL, 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, and 12.5 pg / mL; IL-1β standard at 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.25 pg / mL; and TNF-α standard at 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 15.625 pg / mL. Serum samples do not need to be diluted. Thoroughly mix the reagents and samples during preparation, and minimize foaming.

[0145] b) Set up blank wells, standard wells, and wells for the sample to be tested. Add 100 μL of the standard and sample diluent to the blank wells. Add 100 μL of the standard and serum sample to the remaining wells. Be careful not to create bubbles. Add the sample to the bottom of the plate, avoiding contact with the sides. Gently shake to mix. Cover the plate with film and incubate at 37°C for 90 min.

[0146] c) Discard the liquid in the wells, shake dry, or tap on absorbent paper to dry the liquid in the wells.

[0147] d) Add 100 μL of biotinylated antibody working solution (prepare within 15 minutes before use) to each well, cover with the membrane, and incubate at 37°C for 60 minutes.

[0148] e) Discard the liquid in the wells, spin dry, add 350 μL of washing solution to each well, and wash the plate three times.

[0149] f) Add 100 μL of enzyme conjugate working solution (prepared within 15 minutes before use) to each well, cover with film, and incubate at 37°C for 30 minutes.

[0150] g) Discard the liquid in the wells, spin dry, add 350 μL of washing solution to each well, and wash the plate 5 times.

[0151] h) Add 90 μL of substrate solution (TMB) to each well, cover the plate, and incubate at 37°C in the dark for 15 minutes (this time may be shortened or extended depending on the actual color development, but should not exceed 30 minutes. Terminate when a clear gradient appears in the first four standard wells).

[0152] i) Add 50 μL of stop solution to each well to terminate the reaction. The blue color will immediately turn yellow. The stop solution should be added in the same order as the substrate solution.

[0153] g) Immediately measure the optical density (OD) of each well using a microplate reader at 450 nm. Turn on the microplate reader 15 minutes in advance to preheat the instrument and set the assay program.

[0154] 2.11 Western Blot

[0155] (1) Sample pretreatment

[0156] Rat liver and retinal tissue samples were removed and thawed, and a tissue grinder was pre-chilled. 100 mg of liver and retina, respectively, were weighed and placed in a 2-ml grinding tube. One 4-mm magnetic bead, two 3-mm magnetic beads, and 1 ml of protease lysis buffer (RIPA: protease inhibitor: phosphatase inhibitor = 98:1:1) were added. Tissue grinding parameters were set as follows: liver at 60 Hz, a 30-second run time, a 15-second pause time, three runs, and a set temperature of -10.0°C; retina at 60 Hz, a 30-second run time, a 15-second pause time, six runs, and a set temperature of -10.0°C. The tissue grinding solution was centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, aliquoted, and stored at -80°C until use.

[0157] (2) BCA quantification

[0158] Dilute the retinal protein extract 20 times and the liver protein extract 30 times, take 20 μL of standard or test sample of different concentrations and add them to the corresponding wells; add 200 μL of BCA working solution to each well vertically, cover the well, incubate at 37°C on a shaker for 30 minutes, and measure the OD value at 562 nm using a microplate reader.

[0159] (3) Sample preparation

[0160] Calculate the sample dilution ratio based on the BCA results, with the loading buffer at 1 / 5 of the total volume. Mix thoroughly and heat with a metal domain at 100°C for 10 minutes. Calculate the loading volume based on a loading volume of 30 μg. If the prepared sample is taken out from -20°C, heat it with a thermostatic mixer at 60°C before loading.

[0161] (4) Electrophoresis

[0162] Future PAGE TM (For S100A9, use a 10% Bis-Tris precast gel.) After fixation, add electrophoresis buffer, ensuring that the gel is tight and leak-proof before loading the sample. Place protein ladders on both ends (a 10-180 kDa protein ladder is used for a 10% Bis-Tris precast gel, and a 25-300 kDa protein ladder is used for a 12% Bis-Tris precast gel). After loading, connect the wires (red to red, black to black), set the voltage and time, and run the run (for S100A9, the electrophoresis conditions are 80 V and 90 min). Place the sample back in a -20°C freezer for storage.

[0163] (6) Electroporation

[0164] Select 0.45μmol / L PVDF membrane and activate it with methanol in advance, prepare electrotransfer solution (200ml methanol + 800ml pure water + 3.02g Tris + 14.4g Glycine), cover the PVDF membrane with digital adhesive on the rubber plate, place it between two filter paper-free WB transfer sponges, align and clamp the sample clamps in the transfer tank, place it in the electrotransfer device (black to black), fill it with electrotransfer solution and transfer the membrane in an ice bath (S100A9 electrotransfer conditions are 200mA, 90min).

[0165] (7) Closed

[0166] After the electroporation operation is completed, the adhesive side of the membrane is marked as the front side, and skim milk powder blocking solution (40 mL TBST + 2 g skim milk powder) is added and placed in a 37°C shaker for 1.5 h.

[0167] (8) Apply primary antibody

[0168] Wash with TBST, place in a sealing bag and cut the membrane according to the molecular weight corresponding to the Protein Ladder, add primary antibodies of β-actin (1:8000) and S100A9 (1:1000) diluted with antibody diluent, respectively, place in a 4°C biochemical incubator, incubate overnight at 50-60 speed; if the target protein bands are close together, apply one strip first, wash with Stripping buffer for 15 minutes after development, wash three times with TBST for 5 minutes each time, block, apply primary antibody, etc. as before.

[0169] (9) Apply secondary antibody

[0170] The next day, the primary antibody was recovered (stored at 4°C and can be reused; the specific number of times refers to the development effect), and the sections were washed four times with TBST, each time for 5 min. Secondary antibodies against mouse (1:20,000) and rabbit (1:20,000) diluted in TBST were added and incubated at 37°C for 1 h. The sections were then washed four times with TBST, each for 5 min.

[0171] (10) Development

[0172] Prepare the developer solution according to the ECL kit instructions. Prepare immediately and protect from light. Use a 1:1 ratio of Enhanced Luminescence Solution A to Stabilizer Solution B. Wipe the strips dry with sterile paper and soak in the developer solution. Place the strips in a tray and photograph them using an automated chemiluminescence imaging system. Select Blot - Chemi for the application program, and choose automatic or manual exposure. Select Blot - Colorimetric for the application program, which displays the bright field. After development, return the strips to the TBST solution for storage.

[0173] (11) Statistics

[0174] ImageJ software was used to analyze the grayscale values of protein bands, using β-actin as an internal reference. Normalization was performed in Excel by dividing the grayscale value of the target protein by the grayscale value of the internal reference protein, corresponding to the IntDen values of the target gene and the internal reference.

[0175] 2.12 Targeted Quantification of Acylcarnitines Based on LC-MS / MS

[0176] (1) Sample preparation

[0177] Thaw the sample at 4°C. Accurately weigh approximately 50 mg of sample, homogenize it, add 1000 μL of 75% methanol, and extract with shaking for 1 hour. Place the tube in a refrigerated centrifuge and centrifuge at 12,000 rpm for 10 minutes at 4°C. Add 95 μL of the supernatant to 5 μL of internal standard (C0-D3 500 ng / mL, C2-D3 500 ng / mL, C4-D3 50 ng / mL, C8-D3 50 ng / mL, C16-D3 50 ng / mL) and mix thoroughly. Transfer the solution to a sample vial (containing a 250 μL insert) for mass spectrometry analysis.

[0178] (2) LC-MS / MS detection

[0179] Chromatographic separation was performed using a Waters ACQUITY UPLC I-CLASS ultra-high performance liquid chromatography system under the following conditions: chromatographic column: Waters UPLC HSS T3 (1.7 μm, 2.1 mm × 150 mm); phase A (water, 0.1% formic acid), phase B (acetonitrile:isopropanol 88:12); elution gradient: 95% A (0–0.5 min), 95%–50% A (0.5–2 min), 50%–5% A (2–4 min), 5% A (4–6 min), 5%–95% A (6–6.1 min), 95% B (6.1–8 min); flow rate: 0.26 mL / min, injection volume: 5.0 μL, column temperature: 50 °C

[0180] Mass spectrometry analysis was performed using a Waters XEVO TQ-XS tandem quadrupole mass spectrometer. The positive ion source voltage was 3.0 kV, the cone voltage was 30 V, the ion source temperature was 150°C, the desolvation temperature was 500°C, the desolvation gas flow rate was 950 L / h, and the cone gas flow rate was 10 L / h.

[0181] (3) Data processing

[0182] TargetLynx quantitative software was used to calculate the peak area, and the retention time was allowed to have an error of 15 s. The quantitative results were obtained by the single-point isotope internal standard method.

[0183] 3. Data statistics and analysis

[0184] (1) SPSS 26.0 software was used to perform statistical analysis on the experimental data, and all experiments were repeated more than three times.

[0185] (2) Experimental data were presented as mean ± standard deviation. The t-test was used to compare two groups of experimental data, and one-way analysis of variance was used to compare multiple groups of data. A p-value less than 0.05 was considered statistically significant.

[0186] (3) Use GraphPad Prism 8.0 software to visualize the experimental data.

[0187] 4. Experimental results

[0188] Provide characterization data and effect data, including but not limited to eye symptom scores, liver index, liver function index concentration, ELISA test results, etc.

[0189] 5. Analysis of experimental results

[0190] SPSS 26.0 software was used to perform statistical analysis of the experimental data. All experiments were repeated more than three times, and the experimental data were presented as mean ± standard deviation (mean ± SD). The t-test was used to compare the data between two groups, and the one-way analysis of variance was used to compare the data of multiple groups. A p-value less than 0.05 was considered statistically significant. GraphPad Prism 8.0 software was used to visualize the experimental data.

[0191] 6. Experimental Results

[0192] 6.1 Rat ocular symptoms and scores

[0193] Preganglionic symptoms and clinical scores in rats

[0194] The ocular symptoms of rats in each group were observed using a slit lamp microscope. Figure 2 Compared with the normal group, the rats in the model group showed obvious symptoms such as vasodilation, iris congestion and pupil constriction. Compared with the model group, the eye symptoms of the rats in the dexamethasone group basically returned to normal. The phenolic acids in the panicles, stems, leaves and seeds of Prunella vulgaris improved the symptoms of vasodilation, iris congestion and pupil constriction in the rats.

[0195] The rats in each group were scored according to the clinical scoring standard of uveitis. Figure 3 , Figure 3The eye scores of rats in each group were given; compared with the normal group, **p<0.01, ***p<0.001, ****p<0.0001; compared with the model group, # p < 0.05. As can be seen, compared with the normal group, the ocular scores of rats in the model group were significantly increased; compared with the model group, the ocular scores of rats in the dexamethasone group and the Prunella vulgaris fruit, stem, leaf, and seed phenolic acid groups were significantly reduced. Dexamethasone may increase the burden on the liver, leading to a further increase in liver index, while the phenolic acids in Prunella vulgaris fruit, stem, leaf, and seed groups can effectively improve liver index and have a hepatoprotective effect.

[0196] 6.2 Rat liver index and liver function indicators

[0197] 6.2.1 Rat Liver Index

[0198] The liver index of each group of rats was calculated as follows Figure 4 As shown, Figure 4 is the liver index of rats in each group; compared with the normal group ****p<0.0001; compared with the model group # Compared with the normal group, the liver index of rats in all other groups increased, and the model group showed a significant increase (p < 0.0001). Compared with the model group, the liver index of rats in the positive drug group was higher than that of the model group. After intervention with Prunella vulgaris fruit spike phenolic acid, Prunella vulgaris stem and leaf phenolic acid, and Prunella vulgaris seed phenolic acid, the liver index was significantly lower than that of the model group, with the Prunella vulgaris seed phenolic acid group showing the best effect (p < 0.0001).

[0199] 6.2.2 Rat liver function results

[0200] The AST and ALT levels in the serum of rats in each group were determined by an automatic biochemical analyzer. Figure 5 , Figure 5 The liver function results of rats in each group are shown; compared with the normal group, **p<0.01, ***p<0.001, ****p<0.0001; compared with the model group, #p<0.05. Compared with the normal group, the serum ALT and AST levels of rats in the model group were significantly increased. Compared with the model group, the serum ALT and AST levels of rats in the dexamethasone group and the Prunella vulgaris fruit, stem, leaf, and seed phenolic acid groups were significantly decreased, with the dexamethasone group approaching the normal group. This suggests that the phenolic acids from Prunella vulgaris fruit, stem, leaf, and seed groups can effectively reduce serum ALT and AST levels and improve D-GalN / LPS-induced liver injury.

[0201] 6.3 Histomorphological observation of rats

[0202] 6.3.1 Eye tissue morphology

[0203] HE staining was used to observe the eye tissue morphology of rats in each group. Figure 6 As shown, Figure 6HE staining of the eyes of rats in each group; A, B, C, D, E, and F represent the ciliary body, and G, H, I, J, K, and L represent the retina. A and G represent the normal group, B and H represent the model group, C and I represent the dexamethasone group, D and J represent the Prunella vulgaris fruit acid group, E and K represent the Prunella vulgaris stem and leaf acid group, and F and L represent the Prunella vulgaris seed acid group. Red arrows indicate inflammatory cell infiltration; blue arrows indicate capillary dilation. Compared with the normal group, the model group showed obvious inflammatory infiltration near the ciliary body. Compared with the model group, the dexamethasone group showed no inflammatory infiltration. Prunella vulgaris fruit, stem, leaf, and seed phenolic acids improved the inflammatory infiltration of the ciliary body. This suggests that Prunella vulgaris fruit, stem, leaf, and seed phenolic acids can effectively alleviate D-GalN / LPS-induced anterior chamber inflammation in rat eyes.

[0204] Compared with the normal group, the model group showed retinal structural disorder, with significant folding and vascular inflammation. Compared with the model group, the dexamethasone group showed normal retinal structure and no vascular dilation. The Prunella vulgaris stem and leaf phenolic acid group showed significantly reduced retinal folding, but still showed vascular dilation. The Prunella vulgaris fruit and seed phenolic acid group showed normal retinal structure and alleviated vascular dilation. This suggests that Prunella vulgaris fruit, leaf, and seed phenolic acids can effectively ameliorate D-GalN / LPS-induced retinopathy.

[0205] 6.3.2 Liver tissue morphology

[0206] The results of HE staining of the liver of rats in each group are as follows Figure 7 As shown, Figure 7 HE staining of the livers of rats in each group. The normal group showed regular hepatocyte structure and neat arrangement, neatly distributed hepatic lobules, and clear and regular hepatic cords. Compared with the normal group, the model group showed unrecognizable hepatic cords, irregular hepatocyte shape, deformed or even ablated nuclei, and hepatocyte death. Compared with the model group, the dexamethasone group showed neatly arranged hepatic lobules, regular hepatocyte shape, and no obvious hepatocyte death. The Prunella vulgaris stem and leaf phenolic acid group showed reduced hepatocyte death and a small amount of inflammatory infiltration. The inflammatory infiltration disappeared in the Prunella vulgaris fruit and seed phenolic acid groups, and the liver tissue morphology in the Prunella vulgaris seed phenolic acid group was close to that of the normal group. This indicates that Prunella vulgaris fruit, stem, leaf, and seed phenolic acids can effectively inhibit D-GalN / LPS-induced hepatocyte death and structural disorder, reversing liver damage.

[0207] 6.4 Serum inflammatory factor levels

[0208] The levels of IL-1β, IL-6 and TNFα in the serum of rats in each group were as follows: Figure 8 As shown, Figure 8 Serum ALT and AST levels; compared with the normal group, **p<0.01, ***p<0.001, ****p<0.0001; compared with the model group #p < 0.05. Compared with the normal group, the serum levels of IL-1β, IL-6, and TNFα in the model group rats were significantly increased. Compared with the model group, the serum levels of IL-1β, IL-6, and TNFα in the dexamethasone group were significantly decreased. The serum levels of IL-1β, IL-6, and TNFα in the Prunella vulgaris fruit, stem, leaf, and seed phenolic acid groups were significantly decreased, with the decrease being greater in the Prunella vulgaris seed phenolic acid group. This suggests that the phenolic acids in Prunella vulgaris fruit, stem, leaf, and seed can effectively alleviate the D-GalN / LPS-induced inflammatory response.

[0209] 6.5 Expression of S100A9 in Liver and Retina

[0210] The WB results of S100A9 in the liver and retina of rats in each group are shown in Figure 9 . Figure 9 The WB results of S100A9 in the liver and retina of rats in each group, n=6, compared with the normal group * p<0.05, ** p<0.01; compared with the model group # p < 0.05; ## P < 0.01. Compared with the normal group, S100A9 expression in the liver and retina of rats in the model group was significantly increased. Compared with the model group, S100A9 expression in the liver and retina of rats in the dexamethasone, prunella vulgaris spike acid, prunella vulgaris stem and leaf acid, and prunella vulgaris seed acid groups was significantly decreased. This suggests that prunella vulgaris seed acid can improve D-GalN / LPS-induced liver injury with uveitis in rats by inhibiting S100A9.

[0211] 6.6 Acylcarnitine Content in Liver and Retina

[0212] The acylcarnitine contents in the liver and retina of rats in each group were as follows Figures 10 to 12 As shown, Figure 10 、 Figure 11 and Figure 12 The contents of acylcarnitine in the liver and retina of rats in each group were compared with those in the normal group. * p<0.05, ** p<0.01; compared with the model group # p<0.05, ##p<0.01. C0: Free carnitine; C2: Acetylcarnitine; C3: Propionylcarnitine; C4: Butyrylcarnitine; C5: Isovalerylcarnitine; C6: Hexanoylcarnitine; C8: Heptanoylcarnitine; C8:1: Octenoylcarnitine; C10: Decanoylcarnitine; C10:1: Decenoylcarnitine; C10-0H: Hydroxydecanoylcarnitine; C12: Dodecanoylcarnitine; C12:1: Dodecenoylcarnitine; C12:0-0H: Hydroxydodecanoylcarnitine; C12:1-0H: Hydroxydodecenoylcarnitine; C12-DC: Dodecadienoylcarnitine; C13: Tridecanoylcarnitine; C14: Tetradecanoylcarnitine; C14:1: Tetradecenoylcarnitine; C14:2: Tetradecadienoylcarnitine; C14:0-0H: Hydroxytetradecanoylcarnitine; C14:1 -0H: hydroxytetradecenoylcarnitine; C14:2-0H: hydroxytetradecadienoylcarnitine; C16: hexadecenoylcarnitine; C16:1: hexadecenoylcarnitine; C16:2: tetradecadienoylcarnitine; C16-0H: hydroxyhexadecenoylcarnitine; C16:1-0H: hydroxyhexadecenoylcarnitine; C16:2-0H hydroxyhexadecadienoylcarnitine; C17: heptadecanoylcarnitine; C18: octadecenoylcarnitine; C18:1: octadecenoylcarnitine; C18:2: octadecadienoylcarnitine; C18-0H: hydroxyoctadecenoylcarnitine; C18:1-0H: octadecenoylcarnitine; C18:2-0H: hydroxyoctadecadienoylcarnitine; C20: eicosanoylcarnitine; C20:4: eicosatetraenoylcarnitine. Compared with the normal group, the model group showed a significant increase in acylcarnitine levels in the liver and retina. Compared with the model group, the dexamethasone, prunella vulgaris spike acid, and prunella vulgaris seed phenolic acid groups showed a significant decrease in acylcarnitine levels in the liver and retina. This suggests that prunella vulgaris seed phenolic acid can effectively inhibit D-GalN / LPS-induced acylcarnitine accumulation in the liver and retina.

[0213] In summary, the D-GalN / LPS-induced rat model of liver injury with uveitis showed elevated liver function, significant anterior segment inflammation, and elevated levels of inflammatory factors (IL-6, IL-1β, and TNF-α) in the serum, liver, and retina. Phenolic acids from Prunella vulgaris spikes, stems, leaves, and seeds reduced liver biochemical markers and inflammatory factor levels in the serum, liver, and retina, alleviated ocular inflammatory infiltration, and alleviated liver injury with uveitis. Specifically, phenolic acids from Prunella vulgaris spikes, stems, leaves, and seeds effectively improved D-GalN / LPS-induced liver injury with uveitis, achieving "co-repair" of the liver and eyes. The "co-material basis" is acylcarnitines, and the "co-action mechanism" is that PVPs inhibit S100A9 expression, reduce acylcarnitine accumulation, and suppress inflammatory responses in the liver and retina, resulting in a more effective treatment with Prunella vulgaris seed acids.

[0214] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0215] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. Use of prunella vulgaris phenolic acid in the preparation of products for preventing, treating and / or alleviating liver damage combined with uveitis.

2. The use according to claim 1, characterized in that The prevention, treatment and / or alleviation of liver injury combined with uveitis includes improving the level of IL-6 inflammatory factor.

3. The use according to claim 1, characterized in that The prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of IL-1β inflammatory factor.

4. The use according to claim 1, characterized in that The prevention, treatment and / or alleviation of liver damage combined with uveitis includes improving the level of TNFα inflammatory factor.

5. The use according to any one of claims 1 to 4, characterized in that The dosage forms of the product for preventing, treating and / or alleviating liver damage combined with uveitis include solutions, powders, tablets, granules, capsules, emulsions and suspensions.

6. The use according to claim 5, characterized in that The dosage form of the product for preventing, treating and / or alleviating liver damage combined with uveitis is a solution, mainly composed of prunella vulgaris phenolic acid and a solvent; the solvent includes at least one of dimethyl sulfoxide, polyethylene glycol 300, Tween-80 and normal saline.

7. The use according to claim 6, characterized in that The phenolic acid is derived from at least one of the fruit ears, stems, leaves and seeds of Prunella vulgaris.

8. A drug for preventing, treating and / or alleviating liver damage combined with uveitis, characterized in that: The active ingredient of the drug for preventing, treating and / or alleviating liver damage combined with uveitis includes prunella vulgaris acid.

9. The drug for preventing, treating and / or alleviating liver damage combined with uveitis according to claim 8, characterized in that: The dosage form of the drug for preventing, treating and / or alleviating liver damage combined with uveitis is an oral or injectable dosage form.

10. The drug for preventing, treating and / or alleviating liver damage combined with uveitis according to claim 9, characterized in that: The oral dosage forms include solutions, powders, tablets, granules, capsules, emulsions and suspensions; And or, the injection dosage form includes solution and suspension.

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