Cornus officinalis extract and application thereof in preparation of medicine for treating hepatic fibrosis

Abstract

The invention discloses a dogwood extract and application of the dogwood extract in preparation of a medicine for treating hepatic fibrosis, and belongs to the field of biological medicine. The dogwood extract is obtained by dissolving dogwood powder subjected to alcohol extraction and wine steaming, filtering to obtain filtrate, adding octadecylsilane into the filtrate, mixing, concentrating, performing chromatographic column elution, collecting eluant and drying; the eluent for elution is 50% methanol. Through verification, each active component segment of the wine-steamed dogwood can play a role in resisting hepatic fibrosis by effectively inhibiting inflammation, reversing abnormal collagen deposition, relieving excessive ECM deposition and regulating expression of ECM related factors; in the three active component sections, the effect of the component section MC-50 eluted by 50% of methanol is obviously better than that of the component sections MC-15 and MC-95 eluted by 15% of methanol and 95% of methanol, and dose dependence is achieved. Therefore, the invention provides a new method for clinical treatment of hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the extract of Cornus officinalis and its application in the preparation of drugs for treating liver fibrosis. Background Art

[0002] Liver fibrosis is caused by various reasons that damage hepatocytes, activate hepatic stellate cells, and transform them into myofibroblasts. Inflammatory factors, toxins, etc. stimulate damaged and regenerated hepatocytes, Kupffer cells, sinusoidal endothelial cells, and natural killer (NK) cells to produce immune mediators and cytokines, which act on target cell receptors in an autocrine or paracrine manner, causing myofibroblasts to proliferate and synthesize a large amount of extracellular matrix (ECM). The imbalance between the hyperplasia and degradation of the extracellular matrix leads to excessive deposition of fibrous connective tissue in the liver, disrupting the normal tissue structure of the liver, and thus abnormal liver function. Liver fibrosis is a common pathological process of various chronic liver diseases. Liver fibrosis is reversible, which has prompted a large number of traditional Chinese medicine practitioners to conduct various studies and attempts on the stage of liver fibrosis, aiming to reverse liver fibrosis as much as possible at this stage or in the early stage of cirrhosis to prevent the development to advanced cirrhosis and liver cancer. The phosphatidylinositol-3 kinase / protein kinase (PI3K / Akt) signaling pathway is closely related to the activation, proliferation, apoptosis, and migration of hepatic stellate cells and the secretion of extracellular matrix during the occurrence and development of liver fibrosis. The PI3K / Akt signaling pathway is involved in the occurrence and development of liver fibrosis: the activated PI3K / Akt signaling pathway can promote the formation of liver fibrosis by regulating the proliferation and migration of HSCs and increasing the expression levels of type I and type II collagens; conversely, blocking the activation of the PI3K / Akt signaling pathway can induce HSC apoptosis, promote ECM degradation, and thus delay the process of liver fibrosis development. The main factor for the continuous progression of liver disease patients is the continuous occurrence of inflammation, which further causes the damaged liver to progress towards cirrhosis on the basis of the continuous development of liver fibrosis. Therefore, by regulating the activation of hepatic stellate cells, the production and degradation of liver extracellular matrix, and liver sinusoidal capillarization through the PI3K / Akt signaling pathway, the inflammatory response in the liver can be reduced, thereby exerting an anti-liver fibrosis effect, delaying the disease progression of liver disease patients, and to a certain extent, controlling or even reversing the disease progression of cirrhosis.

[0003] In recent years, Western medicine has made some progress in anti-liver fibrosis treatment targeting various links in the occurrence of liver fibrosis. Many drugs have shown strong anti-fibrosis effects in vitro and animal experiments, but due to the instability of these drugs' efficacy or large toxic and side effects, there are few clinical applications. Currently, only colchicine and interferon-γ (IFN-γ) are applicable clinically and have a certain anti-liver fibrosis effect.

[0004] The therapeutic mechanism of traditional Chinese medicine is relatively complex. It usually acts on multiple targets in the process of liver fibrosis formation and reversal, involves multiple links, and has advantages that Western medicine does not have. After nearly 20 years of research, traditional Chinese medicine plays an anti-liver fibrosis role through inhibiting the generation of collagen fibers and promoting the degradation and absorption of the formed collagen fibers. It has pharmacological effects of multiple components, multiple links, multiple levels, and multiple targets, and has few toxic and side effects, showing obvious advantages in anti-liver fibrosis. Summary of the Invention

[0005] The object of the present invention is to provide the extract of Cornus officinalis and its application in the preparation of drugs for treating liver fibrosis, so as to solve the problems existing in the above-mentioned prior art. The extract of Cornus officinalis MC-50 obtained by eluting with 50% methanol of the present invention has the effect of anti-liver fibrosis, providing a new method for the clinical treatment of liver fibrosis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an extract of Cornus officinalis, which is obtained by dissolving the powder of Cornus officinalis processed by alcohol extraction and wine steaming, filtering to obtain a filtrate, adding octadecylsilane to the filtrate for mixing and concentrating, and then performing column chromatography elution, and collecting the eluate and drying it.

[0008] The eluate for elution is 50% methanol.

[0009] Optionally, the powder of Cornus officinalis processed by alcohol extraction and wine steaming is prepared by the following method:

[0010] Take Cornus officinalis, add yellow rice wine, moisten it for 1 h, steam it at 115 °C for 1 h, and dry it to obtain high-pressure wine-steamed Cornus officinalis;

[0011] Take the high-pressure wine-steamed Cornus officinalis, crush it, add 10 times the amount of 90% ethanol, reflux for 1.5 h, and filter to obtain the first filtrate and the filter residue;

[0012] Add 10 times the amount of 50% ethanol to the filter residue, reflux for 1.5 h, and filter to obtain the second filtrate;

[0013] Combine the first filtrate and the second filtrate, and concentrate and freeze-dry them to obtain;

[0014] The volume ratio of the filtrate to the octadecylsilane is 1:3.

[0015] The present invention also provides the application of the extract of Cornus officinalis in the preparation of drugs for treating liver fibrosis.

[0016] Optionally, the liver fibrosis includes CCl4-induced liver fibrosis.

[0017] Optionally, the extract of Cornus officinalis plays a role in treating liver fibrosis by improving liver damage.

[0018] Optionally, the cornel extract plays a role in treating liver fibrosis by inhibiting excessive deposition of extracellular matrix in the liver cells.

[0019] The present invention also provides a drug for treating liver fibrosis, and the active ingredient is the cornel extract described above.

[0020] Optionally, it is characterized in that the drug further comprises pharmaceutically acceptable excipients.

[0021] Optionally, the excipients include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

[0022] Optionally, the administration mode of the drug includes oral administration or parenteral administration.

[0023] The present invention discloses the following technical effects:

[0024] It has been verified that the present invention finds that each active component segment of alcohol-extracted and wine-steamed cornel can play a role in anti-liver fibrosis. Specifically, it plays a role in anti-liver fibrosis by effectively inhibiting inflammation, reversing abnormal collagen deposition, alleviating excessive deposition of ECM, and regulating the expression of ECM-related factors. And the effect of the active component segment MC-50 eluted with 50% methanol in the three component segments is significantly better than that of the active component segments MC-15 and MC-95 eluted with 15% methanol and 95% methanol, and has a dose-dependence. It can be seen that the present invention provides a new method for the clinical treatment of liver fibrosis. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 It is the yield diagram of each active component segment;

[0027] Figure 2 It is the UPLC diagram of each active component segment; A: The UPLC diagram of MC-15 and alcohol-extracted and wine-steamed cornel; B: The UPLC diagram of MC-50 and alcohol-extracted and wine-steamed cornel; C: The UPLC diagram of MC-95 and alcohol-extracted and wine-steamed cornel;

[0028] Figure 3Evaluation of the improvement effect of each active component segment on CCl4-induced liver injury in mice; A: Animal experiment procedure; B: Serum ALT level; C: Serum AST level; D: Liver photos and H&E staining; E: Liver Masson and Sirius Red staining;

[0029] Figure 4 Evaluation of the inhibitory effect of each active component segment on the excessive deposition of extracellular matrix in CCl4-induced mouse liver; A: Western blot detection results; B: Relative expression level of α-SMA protein in Western blot detection results; C: Relative expression level of CollagenⅠprotein in Western blot detection results; D: Relative expression level of FN protein in Western blot detection results; E: Immunofluorescence detection of the effect of MC-15, MC-50, and MC-95 on α-SMA (200×); F: Immunofluorescence detection of the effect of MC-15, MC-50, and MC-95 on CollagenⅠ(200×); G: Statistical chart of relative expression of α-SMA in immunofluorescence detection results; H: Statistical chart of relative expression of CollagenⅠin immunofluorescence detection results;

[0030] Figure 5 Effect of low / high doses of MC-50 on CCl4-induced liver injury in mice; A: Animal experiment procedure; B: Serum ALT level; C: Serum AST level; D: Liver photos and H&E staining; E: Liver Masson and Sirius Red staining;

[0031] Figure 6 Effect of low / high doses of MC-50 on the excessive deposition of extracellular matrix in CCl4-induced mouse liver; A: Western blot detection results; B: Relative expression level of α-SMA protein in Western blot detection results; C: Relative expression level of CollagenⅠprotein in Western blot detection results; D: Relative expression level of FN protein in Western blot detection results; E: Immunofluorescence detection of the effect of MC-50L, MC-50H, and silymarin on α-SMA (200×); F: Immunofluorescence detection of the effect of MC-50L, MC-50H, and silymarin on CollagenⅠ(200×); G: Statistical chart of relative expression of α-SMA in immunofluorescence detection results; H: Statistical chart of relative expression of CollagenⅠin immunofluorescence detection results. Detailed implementation

[0032] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0037] Example 1 Enrichment of Active Components from Cornus officinalis

[0038] 1. Preparation of Each Component Segment

[0039] 1.1 Sample Pretreatment

[0040] Take the pure Cornus officinalis flesh, add 25% (w / w) of yellow rice wine and moisten it for 1 h, steam it at 115 °C for 1 h, take it out, and dry it at 60 °C to obtain high-pressure wine-steamed Cornus officinalis.

[0041] Take the high-pressure wine-steamed Cornus officinalis, crush it and pass through a No. 2 sieve, add 10 times the amount of 90% ethanol, reflux for 1.5 h, filter through four layers of gauze, add 10 times the amount of 50% ethanol to the filter residue, reflux for 1.5 h, filter, combine the filtrates, concentrate by rotary evaporation, freeze at -80 °C for 24 h, and then lyophilize to obtain the ethanol-extracted and wine-steamed Cornus officinalis powder.

[0042] Take the powder of alcohol-extracted and wine-steamed Cornus officinalis, dissolve it ultrasonically with anhydrous methanol, filter it with filter paper, and then filter it through a 0.22 μm filter membrane. According to the volume ratio of filtrate: ODS = 1:3, weigh the corresponding ODS (octadecylsilane) and mix it with the filtrate, and evaporate and concentrate it to dryness in a water bath at 60 °C.

[0043] 1.2 Medium-pressure preparation

[0044] Load the above sample into the packing column, press the plug with a sieve plate, install the column in sequence, and elute with methanol of different concentrations. The elution program is shown in Table 1; elute 2.0 L of methanol for each active component segment. Chromatographic conditions: chromatographic column Spherical C18 (20 - 45 μm, ); Mobile phase: methanol; Flow rate: 20.0 mL / min.

[0045] Table 1 Elution program

[0046]

[0047] Collect the eluted methanol, and obtain it after rotary evaporation and drying under reduced pressure, named MC-15, MC-50 and MC-95.

[0048] And calculate the yield of each active component segment. Yield = mass of active component segment / mass of alcohol-extracted and high-pressure wine-steamed Cornus officinalis * 100%.

[0049] 2. UPLC detection

[0050] 2.1 Preparation of sample solution

[0051] Weigh the segmented samples MC-15, MC-50 and MC-95 and the alcohol-extracted sample (powder of alcohol-extracted and wine-steamed Cornus officinalis) according to the yield, and make up the volume to a unified volume with methanol. Dilute it to a unified multiple with methanol until the color is clear before use, filter it through a 0.22 μm filter membrane, and inject it for UPLC detection.

[0052] 2.2 Chromatographic conditions

[0053] Chromatographic column 2.1×100 mm Xtimate UPLC C18 (Welch, China); Mobile phase: gradient elution with 0.1% phosphoric acid aqueous solution - acetonitrile (the elution conditions are shown in Table 2), column temperature 30 °C; Flow rate: 0.3 mL / min; Injection volume: 1 μL; Detection wavelength: 240 nm.

[0054] Table 2 UPLC gradient elution program table

[0055]

[0056] 3. Results

[0057] 3.1 Yields of Active Component Segments in Wine-Stewed Cornus officinalis

[0058] Samples of each active component segment were accurately weighed using a balance and the yields were calculated. The yield of MC-15 was 83.57%, the yield of MC-50 was 11.53%, and the yield of MC-95 was 2.26%( Figure 1 ). The results showed that the yield of MC-15 was the highest, and the yield of MC-95 was the lowest, only 2.26%.

[0059] 3.2 UPLC Chromatograms of Active Component Segments in Wine-Stewed Cornus officinalis

[0060] To clarify the distribution of each active component segment in wine-stewed Cornus officinalis (AE), AE and each active segment were detected by UPLC. The results are as Figure 2 shown. The main components are concentrated in the MC-50 segment, a few components overlap with MC-15, and very few components are detected in MC-95.

[0061] Effects of Each Active Component Segment in Example 2 on Liver Fibrosis

[0062] 1. Establishment of Animal Model

[0063] Male BALB / C mice (6 - 8 weeks old, weighing 20 - 22 g) were provided by Zhejiang Vital River Co., Ltd. The experimental animals were housed in the SPF-level barrier system of Zhejiang Chinese Medical University. All mice had free access to food and water. The environmental parameters were maintained at a constant temperature (22 ± 1°C) and relative humidity (50% ± 10%), and a standardized light cycle (12 hours of light / 12 hours of darkness) was uniformly set. The research protocol has been reviewed and approved by the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University, with the license number SYXK(Zhe) 2021 - 0012 and the approval number IACUC - 20250324 - 12.

[0064] A CCl4-induced chronic liver fibrosis mouse model was established. The mice were randomly divided into groups of six each, and the groups were set as: control group, CCl4 model group, and administration groups CCl4 + MC-15, CCl4 + MC-50, and CCl4 + MC-95. Except for the control group injected with ordinary olive oil, the model group and each administration group were subcutaneously injected with 40% CCl4 (the solvent was olive oil) on the back. The dose was doubled in the first week, once in the first week, and then 3 mL / kg, twice a week for a total of 8 weeks( Figure 3 in A). The administration method for the administration groups was intragastric administration. The administration doses, according to the clinical dose and the converted crude drug dose, were MC-15 (2.44 g / kg), MC-50 (0.34 g / kg), MC-95 (0.066 g / kg). The control group and the model group were intragastrically administered an equal amount of 0.25% CMC-Na solution.

[0065] 2. Detection of Blood Biochemical Indexes

[0066] 2.1 Sample collection

[0067] After 6 hours of the last drug administration, all animals were anesthetized by intraperitoneal injection of Zoletil 50, and blood was collected by orbital venous plexus puncture. The blood was collected in 1.5 mL centrifuge tubes, and the mice were dissected to collect the required liver tissues. The blood was allowed to stand at room temperature for 1 hour, centrifuged at 3000 r / min at 4 °C, and the upper serum was taken. The liver tissues were divided and stored in an -80 °C refrigerator and in a sputum cup containing 10% formalin. The sputum cup with formalin can be stored at room temperature for subsequent experiments.

[0068] 2.2 Detection of serum biochemical indicators (ALT, AST)

[0069] The sera of each group were used for detection and analysis (ALT and AST) with a Hitachi 3100 automatic biochemical analyzer.

[0070] 3. Paraffin embedding and sectioning of liver tissues

[0071] The liver tissues stored in the sputum cup with formalin needed to be immersed in the fixative for one week. After fixation, the tissues were taken out, cut into pieces (5 mm × 5 mm × 3 mm), placed in a special embedding cassette, and rinsed with running water for 16 hours to remove the residual formalin in the tissues. The tissue blocks were dehydrated with gradient ethanol, and the process was as follows: 40% ethanol (20 min) - 70% ethanol (15 min) - 80% ethanol (10 min) - 95% ethanol (20 min) - 100% ethanol (30 min), and then transparentized in xylene (30 min), and repeated twice in xylene. The dehydrated tissues were transferred to molten mixed paraffin and stearic acid (1:1), transferred to pure molten paraffin for soaking after 30 min, and then embedded after 40 min. Using a preheated metal embedding mold, the largest cut surface of the tissue was positioned downward, paraffin was injected, and it was cooled to solidify at room temperature, and the wax block was trimmed to make the edge flat.

[0072] The tissue wax blocks to be sectioned were placed in a 4 °C refrigerator overnight in advance to reduce the temperature and enhance the hardness of the paraffin, thereby improving the section quality. Subsequently, the wax blocks were fixed on the stage of a rotary microtome, and rough trimming was carried out (section thickness 20 μm) until the complete tissue section surface was exposed, then changed to 4 μm for formal sectioning and retention. The sections were floated on a constant temperature water bath, and taken on polylysine-coated slides for subsequent experiments.

[0073] 4. H&E staining

[0074] Take 4-μm thick paraffin sections of the liver. After baking at 60 °C for 2 h to melt the wax and enhance adhesion, perform dewaxing treatment with xylene I and II for 10 min each in sequence, followed by gradient ethanol. The process is as follows: 100% ethanol (10 min) - 95% ethanol (5 min) - 90% ethanol (5 min) - 85% ethanol (5 min) - running water for 5 min. Subsequently, stain with hematoxylin solution at room temperature for 3 min, rinse with running water for 5 min, blue with PBS for 2 min and rinse with running water for 5 min. In the counterstaining stage, stain with eosin for 6 min and rinse with running water for 5 min. Dehydrate rapidly with gradient ethanol (sequentially treat with 85%-90%-95%-100% ethanol), clear with xylene for 10 min, then drop neutral resin to cover the slide, and slowly cover the cover glass at a 45° angle to exclude air bubbles. Take the best staining standard as the clear blue staining of liver cell nuclei, the cytoplasmic and fiber gradient pink, and the complete tissue structure without detachment of the section, and perform subsequent microscopic image acquisition operations.

[0075] 5. Masson Staining

[0076] After the tissue sections are dewaxed and gradient hydrated, perform pretreatment according to the H&E staining protocol. Subsequently, stain with modified Weigert iron hematoxylin solution for 8 min, rinse with running distilled water for 1 min, treat with acidic differentiation solution for 10 sec, rinse with running distilled water for 1 min, observe under the microscope until the collagen fibers are light purple, and then treat with buffer blueing solution for 3 min to restore nuclear staining. After staining with Ponceau-fuchsin complex staining solution for 7 min, rinse 3 times with 2% acetic acid aqueous solution, 10 sec each time, differentiate with phosphomolybdic acid solution for 2 min, rinse with acetic acid aqueous solution, and then stain with aniline blue solution for 5 min to label the collagen fibers, and rinse with acetic acid aqueous solution. After staining, the sections are dehydrated with gradient ethanol, cleared with xylene, and sealed with neutral resin, and perform the filming operation under the same conditions as H&E staining.

[0077] 6. Sirius Red Staining (Sirus red)

[0078] Dewax to water routinely according to the item of "H&E staining". Prepare hematoxylin staining solution before use, drop and stain for 5 - 10 min, wash with distilled water for 5 min to remove the excess staining solution, drop and stain with Sirius red staining solution for 15 min, and wash with distilled water for 5 min. After staining, the sections are dehydrated with gradient ethanol, cleared with xylene, and sealed with neutral resin, and perform the filming operation under the same conditions as H&E staining.

[0079] 7. Detection by Western blot method

[0080] 7.1 Extraction of total protein from liver tissue

[0081] Take the liver samples stored at -80°C, weigh about 0.2 mg of tissue blocks and place them in a pre-cooled 1.5 mL centrifuge tube. Add 200 μL of pre-cooled RIPA lysis buffer (containing 1 mM PMSF), and put in a 3 mm diameter stainless steel grinding bead. Load the centrifuge tube onto a cryogenic grinder for grinding. Take it out every 30 seconds to observe whether the homogenate is uniform. If there are still tissue blocks, repeat the operation until the tissue is completely broken into a uniform suspension. Let the homogenate stand on ice for 30 minutes for lysis, and vortex for 15 seconds every 5 minutes during this period to enhance protein dissolution. After lysis is completed, centrifuge at 4°C and 15,000 rpm for 15 minutes, carefully aspirate the supernatant and transfer it to a new pre-cooled centrifuge tube, and measure the protein concentration using the BCA method. The obtained protein samples are aliquoted and stored at -80°C (avoid repeated freezing and thawing), or added with Loading buffer and denatured at high temperature for subsequent Western blot analysis.

[0082] 7.2 Western blot experiment

[0083] (1) Gel preparation

[0084] After rinsing the traceless glass plates with ultrapure water, align and clamp the two glass plates, add ultrapure water and use the water seal method to check for leaks. Prepare a separation gel with an appropriate concentration according to the molecular weight of the target protein, vortex to degas and then inject it into the gap between the glass plates, being careful not to suck in air bubbles, and seal with pure water for 40 minutes. Discard the sealing liquid, use a clean filter paper to absorb the excess pure water, pour in 5% stacking gel, insert a 10 / 15-well sample comb, and polymerize at room temperature for 30 minutes.

[0085] (2) Electrophoresis and membrane transfer

[0086] Install the gel prepared in (1) into the electrophoresis tank and add the diluted 1× electrophoresis buffer. Use a pipette tip to inject the denatured protein sample and protein Marker into the wells. Apply an initial voltage of 80 V to allow the sample to pass through the stacking gel. After the protein Marker reaches the 70 kDa indicator line, increase the voltage to 120 V and continue electrophoresis until the indicator reaches the bottom of the gel. For membrane transfer, use the wet method: Place the transfer tank in an ice-containing foam box and add the pre-diluted 1× transfer buffer. Activate the PVDF membrane with methanol for 30 seconds, assemble the transfer sandwich in the order of the cathode side (black side) [sponge pad - filter paper - gel - PVDF membrane - filter paper - sponge pad], transfer at a constant voltage of 70 V, and maintain an ice bath at 4 - 6°C throughout the process.

[0087] (3) Blocking

[0088] After membrane transfer, immerse the PVDF membrane in 5% non-fat milk - TBST blocking solution and block on a shaker for 1 hour.

[0089] (4) Incubate with primary antibody

[0090] Discard the blocking solution, add an appropriate amount of PBST solution and rinse 3 times, 5 minutes each time. Add the primary antibody diluted according to the titer (diluted with the special primary antibody diluent), and incubate overnight at 4°C with shaking in an incubation bag.

[0091] (5) Incubate the secondary antibody

[0092] Recover the primary antibody the next day, wash the membrane with PBST 3 times (10 minutes each time), and incubate the HRP-labeled secondary antibody at room temperature for 1 hour.

[0093] (6) Exposure detection

[0094] Recover the secondary antibody, wash the membrane with PBST 3 times (10 minutes each time), mix ECL A / B solution at a ratio of 1:1, soak the PVDF membrane for 1 minute, and then collect the signal with a developer. The exposure time is adjusted dynamically according to the signal intensity.

[0095] (7) Membrane regeneration

[0096] After rinsing the PVDF membrane used for the above exposure detection with PBST, shake and elute it with the 1× strong membrane regeneration solution diluted for 30 minutes. After thoroughly rinsing with PBST, it can be reused for the detection of other target proteins. The subsequent steps are the same as (3)-(6).

[0097] 8. Tissue immunofluorescence staining

[0098] Embed the liver tissue stored in the -80°C refrigerator with OCT embedding medium, freeze it at -20°C on the freezing stage of a cryostat. Similar to paraffin sections, cut the sample to a thickness of 4 μm. Use a brush and forceps to assist in sectioning, pick up the sections with polylysine-coated slides to prevent detachment, and place them in the -80°C refrigerator for subsequent experiments. Take out the sections from the -80°C refrigerator for rewarming treatment. After rewarming, immerse the sections in a pre-cooled methanol-acetone mixture at room temperature for 10 minutes. Rinse three times with PBS on a shaker, take out the sections, use a special immunohistochemistry pen to draw a small frame around the sample, add the blocking solution containing 5% goat serum and block at room temperature for 30 minutes. Aspirate it, directly add the primary antibody (diluted with PBS), and incubate in a wet box at 4°C for 16 hours. The next day, recover the primary antibody, rinse three times on a PBS shaker, add the corresponding fluorescent secondary antibody, and incubate in a dark wet box at room temperature for 2 hours. Recover the secondary antibody, rinse three times on a PBS shaker, add the anti-fluorescence quenching solution containing DAPI and cover the slides, then collect images under a fluorescence microscope.

[0099] 9. Results

[0100] 9.1 Evaluation of the improvement effect of each active component segment on CCl4-induced liver injury in mice

[0101] The results are shown in Figure 3, compared with the normal group, the ALT and AST in the model group were significantly increased, indicating that a liver injury model was successfully established. However, there were significant differences in the administration groups. Considering the combined results of ALT and AST, compared with the other two component segments, the MC-50 segment showed the best effect in improving the liver function indicators ALT and AST ( Figure 3 in B-C). By observing the liver morphology, it was found that the surface of the liver in the model group was granular and uneven, the color was dark red and grayish, losing the luster of the normal liver, and lacking elasticity. After administration, the above symptoms were alleviated, and the liver of MC-50 was closest to the normal group. Subsequently, the mouse livers of the three active component segments were paraffin-embedded, sectioned, and pathologically stained, including H&E staining, Masson staining, and Sirus red staining. The tissue liver lobule structure of the normal control group was intact, the hepatocytes were arranged radially, and there was no inflammatory infiltration in the portal area. Masson staining showed only a small amount of collagen deposition in the portal area. The liver fibrosis model group showed significant pathological changes: H&E staining showed that the liver tissue structure was damaged, a large number of inflammatory cells infiltrated around the portal area and central vein, ballooning degeneration of hepatocytes and focal necrosis accompanied by the formation of pseudolobules; Masson staining indicated that collagen was widely deposited in the hepatic sinus and portal area, forming thick fibrous cords and septa; Sirius red staining showed a large amount of red collagen fibers. After the intervention of the drug treatment group, H&E staining showed a reduction in inflammatory infiltration and the arrangement of hepatocytes tended to be regular; Masson and Sirius red staining showed a reduction in the collagen area respectively ( Figure 3 in D-E). The above results confirmed that each active component segment of wine-steamed Cornus officinalis could effectively inhibit inflammation and reverse abnormal collagen deposition, and among the three component segments, MC-50 was superior to MC-15 and MC-95 segments in inhibiting collagen deposition and reducing pathological damage.

[0102] 9.2 Evaluation of the inhibitory effect of each active component segment on the excessive deposition of extracellular matrix in the livers of CCl4-induced mice

[0103] The Western blot results showed that compared with the control group, the protein expressions of α-SMA, CollagenⅠ, and FN in the model group were significantly increased. Compared with the model group, after the intervention of MC-15, MC-50, and MC-95 segments, the expression levels of the related proteins α-SMA, CollagenⅠ, and FN involved in ECM synthesis in the mouse liver could be regulated to varying degrees ( Figure 4 in A-D). The results of immunofluorescence were consistent with those of Western blot. The positive area spots of α-SMA and CollagenⅠ were significantly reduced after the administration of the three component segments, indicating that the administration of the three segments could alleviate the excessive deposition of ECM to varying degrees ( Figure 4In Figures E-H). The above results consistently show that MC-15, MC-50, and MC-95 can play an anti-hepatic fibrosis role by regulating the expression of ECM-related factors, and the effect of MC-50 is better than that of MC-15 and MC-95 segments.

[0104] Study on the Dose-Dependent Anti-Hepatic Fibrosis Effect of the Active Component Segment of MC-50 in Example 3

[0105] 1. Animal Grouping and Treatment

[0106] Male BALB / C mice (6 - 8 weeks old, weighing 20 - 22 g) were provided by Zhejiang Vital River Co., Ltd., with the license SYXK(Zhe)2021 - 0012 and the approval number IACUC - 20250324 - 12. A CCl4-induced chronic hepatic fibrosis mouse model was established. Each group was randomly divided into six mice, and the groups were set as: control group, CCl4 model group, low-dose group CCl4 + MC-50L, high-dose group CCl4 + MC-50H, and positive drug silymarin group. Except for the control group injected with ordinary olive oil, the model and drug administration groups were subcutaneously injected with 40% CCl4 (the solvent was olive oil) on the back. The dose was doubled in the first week, once in the first week, and then 3 mL / kg, twice a week for a total of 8 weeks ( Figure 5 In Figure A). The administration method of the drug administration group was intragastric administration. The administration amounts according to the clinical dose and the converted crude drug amount were MC-50L (0.11 g / kg) and MC-50 (0.34 g / kg); the administration amount of the positive drug silymarin was 100 mg / kg, and the control group and the model group were intragastrically administered an equal amount of CMC-Na solution.

[0107] 2. Detection

[0108] Following the same operations as in Example 2, blood biochemical index detection, paraffin embedding and sectioning of liver tissues, H&E staining, Masson staining, Sirus red staining, Western blot detection, and tissue immunofluorescence staining were performed on each group.

[0109] 3. Experimental Results

[0110] 3.1 Effects of Low / High Doses of MC-50 on CCl4-Induced Liver Injury in Mice

[0111] Through systematic observation, it was found that the livers of mice in the model group presented typical pathological features of fibrosis, manifested as granulation on the liver surface. Pathological staining showed disordered hepatic lobule structure, inflammatory infiltration in the portal area, and abnormal deposition of collagen fibers, etc. After administration of MC-50L, MC-50H, and silymarin respectively, the MC-50H group showed significant therapeutic advantages, with dose-dependence. Serum biochemical tests showed that this component could effectively reverse the abnormal elevation of hepatocyte injury markers ALT and AST ( Figure 5 in B-C). Not only did the MC-50H group restore the normal color and elasticity of the liver in terms of liver morphological improvement, but it also showed outstanding repair ability at the histological pathological level: H&E staining showed that the arrangement of hepatocytes tended to be regular, and the infiltration of inflammatory cells was significantly reduced; double staining with Masson and Sirius red confirmed that the area of collagen deposition was significantly reduced, suggesting its special role in regulating the metabolic homeostasis of ECM ( Figure 5 in D-E).

[0112] 3.2 Effects of low / high doses of MC-50 on excessive deposition of extracellular matrix in the livers of CCl4-induced mice

[0113] The results of Western blot showed that compared with the control group, the protein expressions of α-SMA, CollagenⅠ, and FN in the model group were all significantly increased. Compared with the model group, administration of MC-50H significantly reversed the expressions of the above indicators ( Figure 6 in A-D). The results of immunofluorescence were consistent with those of Western blot. The results showed that MC-50L had a certain ability to relieve the excessive deposition of ECM, and the effect of MC-50H was significantly better than that of MC-50L, with dose-dependence ( Figure 6 in E-H).

[0114] Based on the above experimental results, it was experimentally confirmed that the present invention found that each active component segment of steamed Cornus officinalis could play an anti-hepatic fibrosis role. Specifically, it played an anti-hepatic fibrosis role by effectively inhibiting inflammation, reversing abnormal collagen deposition, relieving excessive deposition of ECM, and regulating the expression of ECM-related factors. Among the three component segments, the active component segment MC-50 eluted with 50% methanol had a better effect than the active component segments MC-15 and MC-95 eluted with 15% methanol and 95% methanol, and had dose-dependence. It can be seen that the present invention provides a new method for the clinical treatment of hepatic fibrosis.

[0115] The above-described embodiments are only used to describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Cornus officinalis extract, characterized in that, The fructus corni extract is obtained by dissolving the ethanol-extracted and wine-steamed fructus corni powder, filtering to obtain a filtrate, adding octadecylsilane to the filtrate for mixing and concentration, followed by column chromatography elution, and collecting and drying the eluate; The eluent for the elution is 50% methanol.

2. The cornel extract according to claim 1, wherein, The ethanol-extracted and wine-steamed fructus corni powder is prepared by the following method: Take fructus corni, add yellow rice wine, moisten for 1 h, steam at 115 °C for 1 h, and dry to obtain high-pressure wine-steamed fructus corni; Take the high-pressure wine-steamed fructus corni, crush it, add 10 times the amount of 90% ethanol, reflux for 1.5 h, and filter to obtain a first filtrate and filter residue; Add 10 times the amount of 50% ethanol to the filter residue, reflux for 1.5 h, and filter to obtain a second filtrate; Combine the first filtrate and the second filtrate, concentrate and freeze-dry to obtain; The volume ratio of the filtrate to the octadecylsilane is 1:

3.

3. Use of the fructus corni extract according to claim 1 in the preparation of a drug for treating liver fibrosis.

4. The application according to claim 3, wherein The liver fibrosis includes CCl4-induced liver fibrosis.

5. The application according to claim 3, characterized in that, The fructus corni extract plays a role in treating liver fibrosis by improving liver injury.

6. The application according to claim 3, characterized in that The fructus corni extract plays a role in treating liver fibrosis by inhibiting excessive deposition of extracellular matrix in the liver.

7. A drug for treating liver fibrosis, characterized in that, The active ingredient is the fructus corni extract according to claim 1.

8. The drug according to claim 7, characterized in that, The drug further includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that, The excipients include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, and flavoring agents.

10. The drug according to claim 7, wherein The administration method of the drug includes oral administration or non-parenteral administration.

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