Application of wild chrysanthemum flower flavone in preparation of product for preventing, treating and / or relieving D-GalN / LPS induced liver and eye injury

Abstract

The invention relates to an application of wild chrysanthemum flower flavone in preparation of a product for preventing, treating and / or relieving D-GalN / LPS induced liver and eye injury. Researchers find that wild chrysanthemum flower flavone can relieve the iris congestion symptom, reduce the content of serum, liver and retina inflammatory factors and relieve eye inflammation infiltration through research, and it is indicated that wild chrysanthemum flower flavone can relieve and treat D-GalN / LPS induced liver and eye injuries. The application provides a new application for wild chrysanthemum flower flavone, and provides a new product for preventing, treating and / or relieving D-GalN / LPS induced liver and eyes. Research finds that when the wild chrysanthemum flower flavone is applied to preparation of products for preventing, treating and / or relieving D-GalN / LPS-induced liver and eye injuries, the drug effect is obvious, the application prospect is wide, and the wild chrysanthemum flower flavone has relatively high popularization and application values.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of wild chrysanthemum flavonoids in the preparation of products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage. Background Art

[0002] The combined use of D-GalN (D-galactosamine) and LPS (lipopolysaccharide) can induce liver and eye damage. This induction method can simulate some clinically similar pathological processes, such as liver cell necrosis and inflammatory response in the liver, corneal opacity, retinal lesions and other injuries in the eyes, thereby affecting the normal functions of related organs.

[0003] At present, there are many limitations in the prevention and treatment of liver and eye damage induced by D-GalN / LPS. For example, traditional anti-inflammatory drugs, such as glucocorticoid drugs (such as dexamethasone, etc.), although they have certain effects in inhibiting inflammatory responses, may bring many side effects if used for a long time or in large quantities. In terms of the liver, it may cause abnormal liver function, such as causing an increase in indicators such as alanine aminotransferase and aspartate aminotransferase, and may also cause fatty liver, liver fibrosis and other lesions. In the eyes, it may increase the risk of eye diseases such as increased intraocular pressure, cataracts, and glaucoma, affecting vision. Traditional prevention and treatment methods have problems such as large side effects, unsatisfactory efficacy, long treatment cycle, and lack of specific and effective preventive measures. Therefore, it is necessary to provide a new drug that can effectively prevent, treat and / or alleviate D-GalN / LPS-induced liver and eye damage. Summary of the invention

[0004] Based on this, the present application provides a new use of chrysanthemum flavonoids, which relates to the use of chrysanthemum flavonoids in the preparation of products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage.

[0005] The specific technical solutions are as follows:

[0006] In a first aspect of the present application, there is provided the use of chrysanthemum flavonoids in the preparation of products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage.

[0007] In some embodiments, the prevention, treatment and / or alleviation of D-GalN / LPS-induced liver and eye damage includes improving the level of IL-6 inflammatory factor.

[0008] In some embodiments, the prevention, treatment and / or alleviation of D-GalN / LPS-induced liver and eye damage includes improving the level of IL-1β inflammatory factor.

[0009] In some embodiments, the prevention, treatment and / or alleviation of D-GalN / LPS-induced liver and eye damage includes improving the level of TNFα inflammatory factor.

[0010] In some of the embodiments, the dosage form of the product for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage is a solution, which is mainly composed of wild chrysanthemum flavonoids and a solvent.

[0011] In some embodiments, the solvent includes at least one of dimethyl sulfoxide, polyethylene glycol 300, Tween-80 and normal saline.

[0012] In some of the embodiments, based on the total mass of the product for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage, the proportion of wild chrysanthemum flavonoids is 30% to 70%.

[0013] This application found through research that wild chrysanthemum flavonoids can relieve iris congestion symptoms, reduce the content of serum, liver and retinal inflammatory factors, and reduce eye inflammatory infiltration, indicating that wild chrysanthemum flavonoids can relieve and treat liver and eye damage induced by D-GalN / LPS. This application provides a new use for wild chrysanthemum flavonoids and provides a new product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS.

[0014] Studies have shown that wild chrysanthemum flavonoids can be used to prepare products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage. The products have obvious efficacy, broad application prospects, and great value for promotion and application.

[0015] The second aspect of the present application also provides a drug for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage, wherein the active ingredient of the drug for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage includes wild chrysanthemum flavonoids.

[0016] In some of the embodiments, the drug for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage is in the form of oral or injectable administration.

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

[0018] And or, the injectable dosage form includes solutions and suspensions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the total ion flow chart of different components of wild chrysanthemum flavonoids in Example 1;

[0020] Figure 2 This is the total ion flow chart of different components of wild chrysanthemum flavonoids in Example 1;

[0021] Figure 3 This is a graph showing the protective effects of different components of wild chrysanthemum flavonoids on liver and eye damage induced by D-GalN / LPS in Example 1;

[0022] Figure 4 This is a graph showing the protective effects of different components of wild chrysanthemum flavonoids on liver and eye damage induced by D-GalN / LPS in Example 1;

[0023] Figure 5 This is a graph showing the protective effect of 70% wild chrysanthemum flavonoids on liver and eye damage induced by D-GalN / LPS in Example 1;

[0024] Figure 6 This is a graph showing the protective effect of 70% wild chrysanthemum flavonoids in Example 1 on liver and eye damage induced by D-GalN / LPS. DETAILED DESCRIPTION

[0025] The application of wild chrysanthemum flavonoids of the present application in the preparation of products for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS is further described in detail below in conjunction with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0026] 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 belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0027] The optional scope of the terms "and / or", "or / and", and "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, said any and all combinations include any combination of any two related listed items, any more related listed items, or all related listed items.

[0028] In this application, "the first aspect", "the second aspect", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "the first", "the second", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0029] In the present 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.

[0030] The weight of the relevant components mentioned in the specification of the examples of this application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the specification of the examples of this application, it is within the scope disclosed in the specification of the examples of this application. Specifically, the weight described in the specification of the examples of this application can be mass units known in the chemical industry such as μg, mg, g, and kg.

[0031] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1-10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0032] 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 the temperature to fluctuate within the accuracy range of instrument control.

[0033] D-galactosamine is an amino sugar. Under normal physiological conditions, the body has a certain metabolic pathway for it. However, when a certain dose of D-GalN is given, it can interfere with certain metabolic processes in liver cells, especially those closely related to the anabolism of proteins and nucleic acids. For example, it can deplete uridine diphosphate-galactose (UDP-Gal) in liver cells, thereby affecting the synthesis of a variety of galactose-containing glycoproteins and glycolipids, resulting in damage to the normal structure and function of liver cells.

[0034] Lipopolysaccharide is the main component of the outer membrane of the cell wall of Gram-negative bacteria and has strong immunogenicity. Once entering the body, LPS can be recognized by the corresponding receptors on the surface of immune cells, activating a series of immune response signaling pathways. LPS can induce inflammatory responses in corneal epithelial cells and stromal cells, prompting these cells to release inflammatory factors such as TNF-α, IL-1β, etc. These inflammatory factors will increase the permeability of corneal epithelial cells, weaken their barrier function, and make them vulnerable to invasion by external pathogens.

[0035] D-GalN / LPS induces liver swelling and hardening. D-GalN interferes with liver cell metabolism and impairs the synthetic function of liver cells. At the same time, inflammatory factors activated by LPS will further increase the burden on liver cells and affect the normal metabolic process of liver cells, such as inhibiting the respiratory function of mitochondria in liver cells, leading to energy metabolism disorders. In histopathological examination, extensive necrosis of liver cells, infiltration of inflammatory cells, and disordered liver lobule structure can be seen. Liver function indicators will also show obvious abnormalities, such as increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, etc., reflecting liver cell damage and impaired liver function. In the cornea, corneal opacity, edema, corneal epithelial cell damage, detachment, etc. can be observed. In the retina, retinal edema, hemorrhage, retinal ganglion cell reduction, degeneration, pigment epithelial cell dysfunction, etc. may occur.

[0036] Through network pharmacology, it was predicted that the possible core genes for wild chrysanthemum flavonoids to regulate liver injury and uveitis are AKT1, BCL2, TP53, HIF1A, and MMP9, which can regulate the expression of inflammatory factors and promote cell repair through the PI3K / AKT and JAK-STAT signaling pathways.

[0037] Based on this, the present application provides the use of wild chrysanthemum flavonoids in the preparation of products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage.

[0038] In some of the embodiments, the method for preparing chrysanthemum flavonoids comprises the following steps S10 to S30.

[0039] Step S10: mixing the wild chrysanthemum sample with the ethanol solution for extraction, separating the solid and the liquid, taking the liquid phase, and obtaining a crude extract containing flavonoids;

[0040] Step S20: adjusting the pH of the crude extract containing flavonoids to 2-6 to obtain a sample solution;

[0041] Step S30: loading the sample solution onto a macroporous resin adsorption column for purification, eluting with an ethanol eluent having a volume concentration of 30% to 70%, collecting the effluent, and obtaining a flavonoid extract.

[0042] In some embodiments, the liquid-to-solid ratio of the ethanol solution to the wild chrysanthemum sample is 18 mL / g.

[0043] In some embodiments, the temperature of the leaching process is 84°C.

[0044] In some of the embodiments, the volume concentration of the ethanol solution in the above-mentioned leaching process is 70%.

[0045] In some embodiments, the leaching treatment time is 100 min.

[0046] In some embodiments, the pH of the crude extract containing flavonoids is 3-4;

[0047] In some embodiments, the loading flow rate of the sample solution is 1 mL / min~5 mL / min.

[0048] In some embodiments, the elution flow rate of the ethanol eluent is 2 mL / min.

[0049] In some embodiments, the concentration of flavonoids in the sample solution is 2.5 mg / mL.

[0050] In some embodiments, the model of the macroporous resin in the macroporous resin adsorption column is AB-8.

[0051] It can be understood that the yield and purity of the wild chrysanthemum flavonoids prepared by the above-mentioned method for preparing wild chrysanthemum flavonoids are high.

[0052] In one specific example, the above-mentioned prevention, treatment and / or alleviation of liver and eye damage induced by D-GalN / LPS includes improving the level of IL-6 inflammatory factor.

[0053] In one specific example, the above-mentioned prevention, treatment and / or alleviation of liver and eye damage induced by D-GalN / LPS includes improving the level of IL-1β inflammatory factor.

[0054] In one specific example, the above-mentioned prevention, treatment and / or alleviation of liver and eye damage induced by D-GalN / LPS includes improving the level of TNFα inflammatory factor.

[0055] In a specific example, the dosage form of the product for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage is a solution, which is mainly composed of wild chrysanthemum flavonoids and a solvent.

[0056] Specifically, the solvent includes at least one of dimethyl sulfoxide, polyethylene glycol 300, Tween-80 and physiological saline, but is not limited thereto.

[0057] In some embodiments, in the above-mentioned product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS, the dosage of the above-mentioned wild chrysanthemum flavonoids is 72 mg / kg to 120 mg / kg. Optionally, in the product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS, the dosage of the above-mentioned wild chrysanthemum flavonoids is 120 mg / kg. It is understood that the specific concentration can be adjusted as needed according to the condition or individual situation.

[0058] In one embodiment, the product further comprises one or more of a binder, a lubricant, a glidant, a diluent and a disintegrant.

[0059] In one embodiment, the binder includes one or more of povidone K30, copovidone, sodium carboxymethylcellulose, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0060] In one embodiment, the lubricant includes one or more of magnesium stearate, talc and polyethylene glycol.

[0061] In one embodiment, the above-mentioned glidant includes one or more of silicon dioxide, micro-powder silica gel, calcium hydrogen phosphate, tricalcium phosphate and milk mineral salt.

[0062] In one embodiment, the diluent includes one or more of microcrystalline cellulose, isomalt, starch, dextrin, lactose, pregelatinized starch, mannitol, sorbitol and erythritol.

[0063] In one embodiment, the disintegrant comprises one or more of cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose and dry starch.

[0064] In some embodiments, the method for preparing the above-mentioned product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS comprises the following steps: mixing wild chrysanthemum flavonoids with a solvent to prepare the above-mentioned product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS. It can be understood that the preparation method can be adjusted as needed according to different dosage forms or formulations.

[0065] In some of the embodiments, based on the total mass of the product for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage, the proportion of the wild chrysanthemum flavonoids is 30% to 70%.

[0066] The present application also provides an animal model for verifying the above-mentioned product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS. The above-mentioned animal model is a liver and eye damage model induced by intraperitoneal injection of 100-200 mg / kg of D-galactosamine and 1 mg / kg of lipopolysaccharide.

[0067] 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.

[0068] The researchers of this application found through research that wild chrysanthemum flavonoids can relieve iris congestion symptoms, reduce the content of serum, liver and retinal inflammatory factors, and reduce ocular inflammatory infiltration, indicating that wild chrysanthemum flavonoids can relieve and treat liver and eye damage induced by D-GalN / LPS. This application provides a new use for wild chrysanthemum flavonoids and provides a new product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS.

[0069] Studies have found that wild chrysanthemum flavonoids can be used to prepare products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye diseases. The products have obvious efficacy, broad application prospects, and great value for promotion and application.

[0070] One embodiment of the present application also provides a drug for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage. The active ingredient of the drug for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage includes wild chrysanthemum flavonoids.

[0071] In some embodiments, the above-mentioned drug for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS is in the form of oral or injection administration.

[0072] In some embodiments, the oral dosage forms include solutions, powders, tablets, granules, capsules, emulsions and suspensions.

[0073] In some embodiments, the above-mentioned injection dosage forms include solutions and suspensions.

[0074] It is understood that the above-mentioned drugs for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS can be administered by subcutaneous injection, intracerebral administration, intravenous injection, oral administration, etc.

[0075] The above-mentioned drugs for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage can reduce liver biochemical indicators, serum, liver and retinal inflammatory factor levels in model rats, reduce eye inflammatory infiltration, and alleviate D-GalN / LPS-induced liver and eye damage.

[0076] The following are specific embodiments

[0077] Unless otherwise specified, the reagents and instruments used in the examples are all conventionally selected in the art. Experimental methods without specific conditions in the examples are carried out according to conventional conditions, such as those described in literature or books or methods recommended by manufacturers.

[0078] Example 1

[0079] 1. Materials

[0080] 1.1 Reagents and instruments

[0081] D-GalN (G115553, Shanghai Aladdin Biochemical Technology Co., Ltd.), LPS (L2880, Sigma-Aldrich®), DMSO (D8371, Beijing Solebold Technology Co., Ltd.), PEG300 (P815612, Shanghai McLean Biochemical Technology Co., Ltd.), Tween-80 (T6336, Shanghai McLean Biochemical Technology Co., Ltd.), normal saline (C23070703, Zhejiang Guojing Pharmaceutical Co., Ltd.), dexamethasone sodium phosphate injection (231101, Shanxi Jinfukang Biopharmaceutical Co., Ltd.), alanine aminotransferase ALT kit (20230707, Shenzhen Raydu Life Sciences Co., Ltd. Company), aspartate aminotransferase AST kit (20230706, Shenzhen Redu Life Science Co., Ltd.), IL-6 (WE05V2J87185, Wuhan Elaruite Biotechnology Co., Ltd.), IL-1β (WE04064B5615, Wuhan Elaruite Biotechnology Co., Ltd.), TNF-α (WE03BB8T3102, Wuhan Elaruite Biotechnology Co., Ltd.), universal cell tissue fixative (20230601, Seven Innovation (Beijing) Biotechnology Co., Ltd.), eyeball fixative (S191109, Wuhan Pinuofei Biotechnology Co., Ltd.).

[0082] Multifunctional microplate reader (BioTek Instruments, Inc.), Chemray 330 fully automatic biochemical detector (Shenzhen Raydu Life Sciences Co., Ltd.), handheld digital slit lamp microscope (KJ5S3, Suzhou Kangjie Medical Co., Ltd.).

[0083] 2. Methods

[0084] 2.1 Preparation of Chrysanthemum flavonoids

[0085] After drying, the wild chrysanthemum medicinal materials were crushed (passed through a 60-mesh sieve), and 70% ethanol was refluxed for 1.5 h, where the liquid-solid ratio of ethanol to the above-mentioned wild chrysanthemum medicinal materials was 18 mL / g, the extraction temperature was 84°C, filtered, and concentrated to an extract concentration of 2.5 mg / mL. AB-8 macroporous resin was used for the enrichment of wild chrysanthemum flavonoids after being soaked in ethanol. The pH of the wild chrysanthemum solution was adjusted to 3-4, the sample volume was 1BV, and the adsorption rate was 2 mL / min. After the solution was fully adsorbed, 30% ethanol, 50% ethanol, and 70% ethanol were used for elution in 3 times the column volume, and the elution rate was controlled to be 2 mL / min. After the eluate was concentrated, it was freeze-dried to obtain three components of wild chrysanthemum flavonoids, with an extraction rate of 1.8% for the 30% component, 2.4% for the 50% component, and 3.0% for the 70% component.

[0086] 2.1.1 Qualitative detection of three components of wild chrysanthemum flavonoids

[0087] 1) Pre-treatment:

[0088] Weigh 50 mg of CF powder accurately into a 2 mL EP tube, add 2 homogenizing grinding beads, add 500 μL of extract (methanol: acetonitrile: water (2:2:1, v / v / v)), the extract contains isotope-labeled internal standard, vortex mix for 30 s, homogenize in a homogenizer (35 Hz, 240 s), ultrasonicate in an ice-water bath for 5 min, and repeat the homogenization and ultrasonication steps 3 times. After the ultrasonication, the sample solution was allowed to stand at -40°C for 30 min, then centrifuged at 4°C for 15 min (12000 rpm), and the supernatant was taken. The supernatant was allowed to stand at -40°C for 10 min, then centrifuged at 4°C for 15 min (12000 rpm), and the supernatant was filtered through a 0.22 μm microporous membrane into a sample vial for detection.

[0089] 2) Detection method:

[0090] Liquid chromatography conditions: Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Scientific), Phenomenex Kinetex C18 column (2.1×100 mm, 2.6 μm), column temperature 30 °C, detection wavelength 334 nm, flow rate 0.3 mL / min, injection volume 2 μL. Isopropanol:acetonitrile (1:1, V / V) (A)-0.01% acetic acid water (B) was used as the mobile phase, and the gradient elution program (A) was set as: 1%-1% (0-1 min), 1%-99% (1-8 min), 99%-99% (8-9 min), 99%-1% (9-9.1 min), 1%-1% (9.1-12 min).

[0091] Mass spectrometry conditions: Orbitrap Exploris 120 mass spectrometer (Thermo), sheath gas flow: 50Arb, auxiliary gas flow: 15Arb, capillary temperature: 320°C, full millisecond resolution: 60000, MS / MS resolution: 15000, collision energy: SNCE 20 / 30 / 40, spray voltage: 3.8 kV (positive ion) or -3.4 kV (negative ion).

[0092] 3) Results

[0093] The total ion flow chart of 30%, 50%, and 70% components of total flavonoids from wild chrysanthemum is as follows Figure 1~Figure 2 The qualitative results of the components are shown in Table 1 below.

[0094] Table 1

[0095]

[0096]

[0097] Among them, the 30% component contains the following ingredients: Quinic acid, Neochlorogenicacid, Chlorogenic acid, Cryptochlorogenic acid, Caffeicacid, Luteolin 7-rutinoside, 1,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Di-O-caffeoylquinic acid methyl ester and Isochlorogenic acid A.

[0098] The 50% component contains the following ingredients: Quinic acid, Neochlorogenic acid, Chlorogenic acid, Cryptochlorogenic acid, Apigenin-7-O-glucoside, Caffeic acid, Luteolin 7-rutinoside, Nicotiflorin, Luteolin 7-O-glucuronide, 1,4-Dicaffeoylquinic acid, Diosmin, Rhoifolin, Apigenin-7-O-rutinoside, Kaempferol 7-O-glucoside, 6-O-Methylscutellarin, Diosmetin-7-O-β-D-glucopyranoside, 4,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, Apigenin-7-O-glucuronide, Isorhamnetin-3-O-glucoside, 4,5-Di-O-caffeoylquinic acid methyl ester, Isochlorogenic acid A, Linarin, Luteolin 7-O-glucoside.

[0099] The 70% component contains the following ingredients: Apigenin-7-O-glucoside, Nicotiflorin, Luteolin 7-O-glucuronide, 1,4-Dicaffeoylquinic acid, Diosmin, Rhoifolin, Apigenin-7-O-rutinoside, Kaempferol 7-O-glucoside, 6-O-Methylscutellarin, Diosmetin-7-O-β-D-glucopyranoside, 4,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, Apigenin-7-O-glucuronide, Isorhamnetin-3-O-glucoside, 4,5-Di-O-caffeoylquinic acid methyl ester, AIsochlorogenic acid A. Acacetin, Linarin, Eupatilin, Luteolin 7-O-glucoside, Eriodictyol, Quercetin, Isorhamnetin, Apigenin, Kaempferol, Tricin, Luteolin, Isoquercitrin and Cirsiliol.

[0100] 2.2 Animal medication and modeling

[0101] 5-6 week old SD rats were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (Batch No.: DWDG-202311130014). All rats were individually housed at a constant temperature (24°C) with a light / dark cycle of 12 hours. After one week of adaptive feeding, they were randomly divided into a normal group (CON), a model group (MOD), a dexamethasone group (DXMS), a 30% group (30%, 72 mg / kg), a 50% group (50%, 96 mg / kg), and a 70% group (70%, 120 mg / kg). After screening out the best efficacy of the above three components, low-dose (L) and high-dose (H) groups were set up to further verify the efficacy (the ratio of low, medium, and high doses was 1:3:9). Twelve rats were set up in each group. Specifically, the adult dosage of wild chrysanthemum specified in the Chinese Pharmacopoeia is 9-15 g, and 15 g / person is selected as the low dose. The conversion for rats is as follows: 15 g / 70 (Kg*human)*6.3= 1.35 g / (kg*rat).

[0102] According to the calculation of low dose: medium dose: high dose = 1:3:9, the medium dose of each component is:

[0103] 30% component dosage: 1.35 * 3 * 1.8% = 72 mg / kg;

[0104] 50% component dosage: 1.35 * 3 * 2.4% = 96 mg / kg;

[0105] 70% component dosage: 1.35 * 3 * 3.0% = 120 mg / kg;

[0106] Therefore, the 70% component low dose is 40 mg / kg and the 70% component high dose is 360 mg / kg.

[0107] Rats were given preventive administration of various components of wild chrysanthemum flavonoids for one week (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline as solvent), and the dexamethasone group was given a single dose before modeling (1 mg / kg). All rats were fasted for 12 h before modeling and modeled 2 h after administration (dose of 150 mg / kg D-GalN + 1 mg / kg LPS, normal saline as solvent). After 18 h, slit lamp images of the eyes were taken and anesthetized. Blood was collected from the abdominal aorta, serum was separated, liver was collected, retina was peeled off, stored at -80 °C, and used. The experiment was carried out in accordance with the animal research protocol approved by the Ethics Committee of Hunan University of Chinese Medicine (SLBH-202309140005).

[0108] 2.3 Liver function tests

[0109] Serum samples from each group were loaded onto the instrument, and ALT and AST were tested according to the instructions.

[0110] 2.4 Eye inflammation score

[0111] Ocular symptoms were observed under a slit lamp microscope, and the rats' iris congestion, pupil constriction, anterior chamber exudation, and anterior chamber abscess were scored.

[0112] Table 2 Clinical scores of uveitis in rats

[0113]

[0114] 2.5 Histomorphology

[0115] The liver and eyeballs were removed from rats in each group. The liver was fixed with tissue fixative, and the eyeball was fixed with eyeball fixative, embedded in paraffin, sliced, stained with hematoxylin and eosin, and the stained area was observed under an optical microscope. The liver damage was scored pathologically, including hepatocyte necrosis, hepatocyte edema, and inflammatory cell infiltration. The scoring criteria were: 0, none; 1, mild; 2, moderate; 3, severe, and the final score was the sum of the scores of the above three items. The ciliary inflammation was observed, and the integrity of the cells in each layer of the retina, the neatness of the cell arrangement, and the regularity of the nerve fiber layer were observed.

[0116] 2.6 Detection of inflammatory factors

[0117] Take 100 mg of liver sample and 60 mg of retinal sample, add 1 ml of protease lysis solution (PBS: protease inhibitor = 99:1), grind thoroughly (liver sample grinding frequency 30s / time, run times 2 times; retinal sample running frequency 45s / time, run times 6 times), centrifuge (15000r / min, 10min), take the supernatant, and package. Rat serum, liver, and retinal samples were operated according to the instructions of the kit and tested on the machine.

[0118] 2.7 Data Analysis

[0119] The data results were analyzed by SPSS 21.0 statistical software. One-way ANOVA was used for comparison among multiple groups, and t-test was used for comparison among groups. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0120] 3. Experimental Results

[0121] 3.1 Screening of the best efficacy of different components of wild chrysanthemum

[0122] Figure 3The protective effects of different components of wild chrysanthemum on liver and eyes induced by D-GalN / LPS (A: biochemical indexes, B: liver HE staining, C: eye inflammation under slit lamp, D: ciliary body HE staining, E: retinal HE staining). Figure 4 The protective effects of different components of wild chrysanthemum on liver and eyes induced by D-GalN / LPS (A: IL-6 levels in serum and tissues, B: IL-1β levels in serum and tissues, C: TNF-α levels in serum and tissues). After intraperitoneal injection of D-GalN / LPS in rats, serum ALT and AST were significantly upregulated. The three components of wild chrysanthemum could significantly reduce the ALT level (p<0.05), and the 70% component had the best effect (p<0.05) ( Figure 3 A) in the figure is shown in Table 3.

[0123] Table 3

[0124]

[0125] HE staining was used to observe the morphological changes of rat liver tissues, and it was found that D-GalN / LPS caused focal inflammatory infiltration in rat liver tissues (black arrows), focal necrosis of hepatocytes in many places, and vacuolation of liver lobules (blue arrows), indicating that D-GalN / LPS caused obvious liver damage. After drug intervention, the degree of inflammatory infiltration and cell necrosis in the liver tissues of rats in the 30% component, 50% component, and 70% component groups was significantly reduced, and the liver cords of rats in the 70% component group were clearly distinguished ( Figure 3 B) is specifically shown in Table 4.

[0126] Table 4

[0127]

[0128] The slit lamp microscope was used to examine the eye condition of the rats, and it was found that D-GalN / LPS could cause obvious iris congestion and pupil constriction in the rats. Compared with the model group, the congestion symptoms in the drug group were alleviated to a certain extent ( Figure 3 C) is specifically shown in Table 5.

[0129] Table 5

[0130]

[0131] To illustrate the morphological changes of the eyes, HE staining of ciliary body and retinal tissues was observed. Compared with normal rats, the ciliary body of the model rats had obvious inflammatory infiltration ( Figure 3 D in the figure), retinal ganglion cell vacuolization and loss (black arrows), and irregular nerve fiber layer (blue arrows) ( Figure 3 (E) After 70% component intervention, the pathological features were significantly improved.

[0132] ELISA assay showed that D-GalN / LPS significantly increased the levels of IL-6, IL-1β and TNFα in serum, liver and retina ( Figure 4 AC in the sample), indicating that D-GalN / LPS can cause inflammatory responses in the liver and retina. Analysis of the pharmacodynamic effects of the three components of wild chrysanthemum showed that the 50% component and the 70% component could significantly reduce the levels of IL-6 and IL-1β in serum (p<0.05), but had no statistical significance on the level of serum TNF-α. All three components could significantly improve the content of IL-6, IL-1β and TNFα in the liver (p<0.05), and the efficacy of the 50% component and the 70% component was better than that of the 30% component (p<0.05). Compared with the 30% component and the 50% component, the 70% component could significantly improve the level of inflammatory factors in the retina (p<0.05), as shown in Tables 6 to 8.

[0133] Table 6

[0134]

[0135] Table 7

[0136]

[0137] Table 8

[0138]

[0139] In summary, compared with the 30% and 50% components, the 70% component of wild chrysanthemum has the best efficacy and can effectively alleviate D-GalN / LPS-induced liver and eye damage.

[0140] 3.2 Protective effect of 70% wild chrysanthemum components on D-GalN / LPS-induced liver and eye damage in rats

[0141] Figure 5 The protective effect of 70% wild chrysanthemum components on liver and eyes induced by D-GalN / LPS (A: biochemical indexes, B: liver HE staining, C: eye inflammation under slit lamp, D: ciliary body HE staining, E: retinal HE staining). Figure 6 The protective effect of 70% wild chrysanthemum on liver and eyes induced by D-GalN / LPS (A: IL-6 levels in serum and tissues, B: IL-1β levels in serum and tissues, C: TNF-α levels in serum and tissues). After intraperitoneal injection of D-GalN / LPS in rats, serum ALT and AST were significantly upregulated. Low, medium and high doses of 70% wild chrysanthemum could significantly downregulate liver biochemical indicators (p<0.05), and the efficacy of the medium and high dose groups was better than that of the low dose group (p<0.05) ( Figure 5 A) in the figure is shown in Table 9.

[0142] Table 9

[0143]

[0144] Observation of the results of HE staining of the liver of rats in each group showed that compared with the model group, the inflammatory infiltration (black arrow) and hepatocyte necrosis (blue arrow) in the drug group were significantly improved ( Figure 5 B) is specifically shown in Table 10.

[0145] Table 10

[0146]

[0147] The slit lamp microscope was used to examine the eye condition of the rats, and it was found that the irises of the model rats were congested and the pupils were constricted, while the congestion symptoms of the drug group were alleviated to a certain extent ( Figure 5 C) is specifically shown in Table 11.

[0148] Table 11

[0149]

[0150] Analysis of the HE staining results of the retinal tissue and ciliary body of rats in each group showed that the vacuolation of retinal ganglion cells in the model group was aggravated (black arrows), and the regularity of the nerve fiber layer was uneven (blue arrows) ( Figure 5 D in the figure), there is a large amount of inflammatory cell infiltration in the ciliary body, and each dose of the 70% component has a certain improvement effect.

[0151] The levels of IL-6, IL-1β and TNFα in serum, liver and retina of rats in each group were analyzed ( Figure 6 AC in the model group), as shown in Tables 12 to 14, the levels of inflammatory factors in the serum, liver and retina of rats in the model group were significantly increased, and the medium and high dose groups of 70% component could significantly reduce the levels of inflammatory factors in the serum, liver and retina of rats (p<0.05).

[0152] Table 12

[0153]

[0154] Table 13

[0155]

[0156] Table 14

[0157]

[0158] In summary, in the D-GalN / LPS-induced rat liver and eye injury model, the model rats had increased liver function, obvious anterior segment inflammation, and increased levels of inflammatory factors (IL-6, IL-1β, TNF-α) in serum, liver, and retina. The 30%, 50%, and 70% components of total flavonoids from wild chrysanthemum can reduce liver biochemical indicators, serum, liver, and retinal inflammatory factor levels in model rats, reduce ocular inflammatory infiltration, and alleviate D-GalN / LPS-induced liver and eye damage, and the 70% component has the best effect.

[0159] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0160] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it 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 modifications and improvements can be made, which all belong to 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 of this application shall be based on the contents of the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. Application of wild chrysanthemum flavonoids in the preparation of products for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage.

2. The use according to claim 1, characterized in that: The prevention, treatment and / or alleviation of liver and eye damage induced by D-GalN / LPS 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 and eye damage induced by D-GalN / LPS 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 and eye damage induced by D-GalN / LPS 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 form of the product for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS is a solution, which is mainly composed of wild chrysanthemum flavonoids and a solvent.

6. The use according to claim 5, characterized in that: The solvent includes at least one of dimethyl sulfoxide, polyethylene glycol 300, Tween-80 and physiological saline.

7. The use according to claim 6, characterized in that: Based on the total weight of the product for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage, the proportion of wild chrysanthemum flavonoids is 30% to 70%.

8. A drug for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS, characterized in that: The active ingredients of the drug for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS include wild chrysanthemum flavonoids.

9. The drug for preventing, treating and / or alleviating D-GalN / LPS-induced liver and eye damage according to claim 8, characterized in that: The dosage form of the drug for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS is an oral or injection dosage form.

10. The drug for preventing, treating and / or alleviating liver and eye damage induced by D-GalN / LPS according to claim 9, characterized in that: The oral dosage forms include solutions, powders, tablets, granules, capsules, emulsions and suspensions; And or, the injectable dosage form includes solutions and suspensions.

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