Application of semiliquidambar cathayensis in preparation of medicine for preventing and treating liver diseases

Abstract

The invention discloses application of semiliquidambar cathayensis in preparation of a medicine for preventing and treating liver diseases, and belongs to the technical field of traditional Chinese medicines. The application refers to application of the semiliquidambar cathayensis, the extract thereof or the pharmaceutical composition thereof in preparation of drugs for preventing and treating hepatic fibrosis, drugs for preventing and treating hepatic diseases by improving hepatic fibrosis and drugs for preventing and treating hepatic diseases. Experiments prove that the semiliquidambar cathayensis has a good treatment effect on liver fibrosis, and a basis is provided for clinical medication conversion of the semiliquidambar cathayensis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and relates to the application of Semiliquidambar cathayensis H.T.Chang. in the preparation of drugs for preventing and treating liver diseases, specifically to the application of Semiliquidambar cathayensis H.T.Chang., its extract or its pharmaceutical composition in the preparation of drugs for preventing and treating liver diseases. Background Art

[0002] Liver fibrosis refers to a wound healing response that occurs in the body when various pathogenic factors cause chronic liver injury, mainly characterized by excessive collagen accumulation. Liver fibrosis is a chronic and reversible pathological phenomenon. If not treated in time, it may develop into irreversible liver cancer or cirrhosis. Western medicine treats liver fibrosis mainly by antiviral and symptomatic treatment, but it has disadvantages such as few types of treatment drugs and many toxic and side effects. Therefore, it is of great significance to explore new methods for treating liver fibrosis.

[0003] Semiliquidambar cathayensis H.T.Chang. is a plant of the genus Semiliquidambar in the family Hamamelidaceae. It has the effects of dispelling wind and dampness, relaxing tendons and activating blood circulation, and can be used to treat rheumatic arthritis, traumatic injury, postpartum wind paralysis and other diseases. Modern pharmacological studies have shown that Semiliquidambar cathayensis H.T.Chang. has the effects of analgesia, anti - inflammation, antioxidant and promoting blood circulation to remove blood stasis.

[0004] Currently, the research on Semiliquidambar cathayensis H.T.Chang. in the liver aspect is all about using it in combination with traditional Chinese medicines with liver - protecting effects, and there is a lack of corresponding mechanism and effect verification research, making it difficult to measure its effectiveness. For example, CN106822434A and CN106822426 disclose that the combination of Semiliquidambar cathayensis H.T.Chang. with snake, Ligusticum wallichii, Chaenomeles sinensis, Homalomena occulta, Aconitum carmichaelii var. wilmianum, Gentiana macrophylla, Cyathula officinalis, etc. can treat hepatitis A and hepatitis B. CN103550513A discloses that the combination of Semiliquidambar cathayensis H.T.Chang. with Piper cubeba, Elsholtzia ciliata, Clintonia udensis, Wikstroemia glabra, Diplochne fusca, Picea asperata Mast., Dendrophthoe glabrescens, Semiliquidambar cathayensis H.T.Chang., Oldenlandia auricularia, Herba Lysimachiae, Tamarindus indica, Coriaria nepalensis, Gendarussa vulgaris, Combretum alfredii, Hemiboea subcapitata, Codonopsis convolvulacea and Wikstroemia chamaedaphne can treat cholestatic hepatitis. CN103356886A discloses that the combination of Semiliquidambar cathayensis H.T.Chang. with Rosa laevigata, Scrophularia ningpoensis, Euphorbia jolkinii, Desmodium racemosum, Entada phaseoloides, Mosla chinensis, Michelia champaca, Bupleurum chinense, Cassia obtusifolia, Cyperus rotundus, Hibiscus trionum, Podophyllum hexandrum, Osbeckia chinensis, Lysimachia parviflora, Pilea microphylla, Vitis bryoniifolia and Gynura divaricata can treat primary biliary cirrhosis.

[0005] CN118021857A discloses the application of Semiliquidambar cathayensis H.T.Chang. in the preparation of drugs for preventing and / or treating liver injury. It proves that Semiliquidambar cathayensis H.T.Chang. has good preventive and therapeutic effects on acute chemical liver injury, drug - induced liver injury and alcoholic liver injury by using an acute liver injury model. This invention does not disclose the influence of Semiliquidambar cathayensis H.T.Chang. on liver fibrosis. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of Radix Angelicae Pubescentis in the preparation of a medicament for preventing and treating liver diseases; in particular, the use of Radix Angelicae Pubescentis, its extract or its pharmaceutical composition in the preparation of a medicament for preventing and treating liver diseases. The present invention finds that Radix Angelicae Pubescentis can effectively reduce the progression of liver fibrosis, providing experimental basis and scientific support for the potential application of Radix Angelicae Pubescentis in the treatment of liver diseases.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of Radix Angelicae Pubescentis, an extract thereof or a pharmaceutical composition thereof in the preparation of a medicament for preventing and treating liver fibrosis.

[0009] In a second aspect, the present invention provides the use of Radix Angelicae Pubescentis, its extract or its pharmaceutical composition in the preparation of a medicament for preventing and treating liver diseases by improving liver fibrosis.

[0010] In a third aspect, the present invention provides the use of Radix Angelicae Pubescentis, its extract or its pharmaceutical composition in the preparation of a medicament for preventing and treating liver diseases.

[0011] In some embodiments, the liver disease comprises at least one of non-alcoholic fatty liver disease, alcoholic fatty liver disease, liver cysts, benign tumors, hepatitis, and cirrhosis.

[0012] The Hamamelidaceae of the present invention is derived from Semiliquidambarcathayensis HTChang. of the Hamamelidaceae family. Preferably, the Semiliquidambarcathayensis HTChang is the above-ground part and / or underground part of the Semiliquidambarcathayensis. Further preferably, the above-ground part includes stems and / or branches, and the underground part includes roots and / or rhizomes.

[0013] The Herba Lysimachiae extract of the present invention is Herba Lysimachiae water extract and / or alcohol extract.

[0014] In some embodiments, the preparation method of the Herba Lycopodiellae alcohol extract is as follows: adding Herba Lycopodiellae to alcohol for reflux extraction, and performing solid-liquid separation to obtain the Herba Lycopodiellae alcohol extract.

[0015] In some specific embodiments, the volume fraction of the alcohol is 10-90%, and the amount of the alcohol added is 5-30 times the weight of the golden vine half maple; the extraction is performed at least once, each time for 0.5-3 hours.

[0016] Preferably, the volume fraction of the alcohol is 20-80%; more preferably 30-75%; further preferably 50-75%; most preferably 70%.

[0017] Preferably, the amount of the alcohol added is 8-20 times the weight of the Herba Lycopodii; more preferably 8-15 times.

[0018] Preferably, the extraction is performed 1-5 times, each time for 0.5-2 hours; further preferably, the extraction is performed 1-3 times, each time for 1.5 hours.

[0019] In some specific embodiments, the alcohol is ethanol and / or methanol; preferably ethanol.

[0020] In some embodiments, the preparation method of the water extract of Radix Angelicae Pubescentis is as follows: adding Radix Angelicae Pubescentis to water for reflux extraction, and performing solid-liquid separation to obtain the extract.

[0021] In some specific embodiments, the amount of water added is 5-30 times the weight of the Herba Lycopodii; and the extraction is performed at least once, each time for 0.5-3 hours.

[0022] The extract of Radix Angelicae Pubescentis of the present invention can also be processed by any one of concentration, enrichment and purification, preferably concentration.

[0023] In a third aspect, the present invention provides a pharmaceutical composition, which is prepared from the aforementioned Herba Lycopodii or its extract and pharmaceutically acceptable excipients.

[0024] In some specific embodiments, the pharmaceutical composition is in the form of granules, tablets, capsules, powders, pills, sprays or liquid preparations.

[0025] The beneficial effects of the present invention are:

[0026] (1) Experiments have shown that the Herba Lycopodii of the present invention has a good therapeutic effect on liver fibrosis, which provides a basis for the transformation of Herba Lycopodii into clinical medication.

[0027] (2) The present invention is the first to discover that Herba Lycopodii has a good therapeutic effect on liver fibrosis, providing a new approach for the treatment of liver diseases and, at the same time, expanding the application field of Herba Lycopodii. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The experimental design flow chart of Example 2 is shown below;

[0029] Figure 2 This is a line graph showing changes in rat body weight in Example 2;

[0030] Figure 3 This is the last weight gain graph of rats in Example 2. Compared with the Con group, # Indicates P < 0.001; compared with the Mod group, * indicates P < 0.05, ** indicates P < 0.01;

[0031] Figure 4 This is the macroscopic morphology of the rat liver in Example 2;

[0032] Figure 5 The liver weight and liver index of rats in Example 2, where A is the liver weight of rats and B is the liver index. Compared with the Con group, ### Indicates P < 0.001; compared with the Mod group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001;

[0033] Figure 6 The serum liver function levels of rats in Example 2, where A is the weekly change trend of serum ALT in rats; B is the weekly change trend of serum AST in rats. Compared with the Con group, ### indicates P < 0.001; *** indicates P < 0.001 compared with the Mod group;

[0034] Figure 7 The serum liver function levels of rats in Example 2, where A is the serum ALT level of rats; B is the serum AST level of rats. Compared with the Con group, ### indicates P < 0.001; *** indicates P < 0.001 compared with the Mod group;

[0035] Figure 8 The staining results of rat pathological sections in Example 2, A is the Sirius red staining section of rat liver tissue pathology, B is the statistics of Sirius red staining results of liver (4x, scale = 200 μm), compared with the Con group, ### indicates P < 0.001; *** indicates P < 0.001 compared with the Mod group;

[0036] Fig. 9 The staining results of rat pathological sections in Example 2, A is the Masson staining section of rat liver tissue pathology; B is the statistics of Masson staining results of liver (4x, scale = 200 μm), compared with the Con group, ### indicates P < 0.001; *** indicates P < 0.001 compared with the Mod group;

[0037] Fig.10 This is the liver tissue lesion score diagram of Example 2, A is the Ishake score of rats, B is the percentage of fatty degeneration, and C is the statistical result of the percentage of ballooning degeneration. Compared with the Con group, ### indicates P < 0.001; *** indicates P < 0.001 compared with the Mod group;

[0038] Fig.11 The experimental design flow chart of Example 3 is shown below;

[0039] Fig.12This is the macroscopic morphology of the mouse liver in Example 3;

[0040] Fig.13 The serum liver function levels of mice in Example 3, where A is the weekly change trend of serum ALT in rats; B is the weekly change trend of serum AST in rats. Compared with the Con group, ### indicates P < 0.001; *** indicates P < 0.001 compared with the Mod group; Fig.14 This is the Masson staining section of mouse liver tissue pathology in Example 3. DETAILED DESCRIPTION

[0041] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0042] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0043] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0044] The purpose of "solid-liquid separation" described in the present invention is to achieve separation of solid phase and liquid phase, which can be achieved by conventional technical means, such as filtration, centrifugation, filter pressing or separation and purification, preferably filtration.

[0045] The "pharmaceutical composition" described in the present invention comprises Herba Lycopodii or its extract and pharmaceutically acceptable excipients. In a specific embodiment, Herba Lycopodii or its extract described in the present invention is provided in the pharmaceutical composition in an effective amount (e.g., a therapeutically effective amount).

[0046] The "pharmaceutically acceptable" ingredients described in the present invention are substances that are suitable for human beings and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The "pharmaceutically acceptable excipients" include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients (such as cocoa butter and suppository wax), colorants, coating agents, sweeteners and flavoring agents may also be present in the pharmaceutical composition.

[0047] The "pharmaceutical composition" and "drug" described in the present invention can be prepared by any method known in pharmacy. Generally speaking, these preparation methods include associating the Herba Artemisiae Argyi or its extract (i.e., the only active ingredient) with a carrier or excipient and / or one or more other auxiliary ingredients, and then if necessary and / or desired, molding and / or packaging the product into a desired single dose or multiple dose unit.

[0048] The pharmaceutical compositions and drugs of the present invention can be prepared according to known methods, such as the methods described in the general rules for preparation of the Chinese Pharmacopoeia 2020, the Japanese Pharmacopoeia 16th edition, the United States Pharmacopoeia, and the European Pharmacopoeia 9th edition. Depending on the dosage form, the pharmaceutical compositions and drugs of the present invention can be appropriately administered to patients.

[0049] The sole active ingredient, pharmaceutically acceptable excipient in the "pharmaceutical composition" described herein will vary depending on the identity, size and / or condition of the subject being treated and further on the route of administration of the composition. The pharmaceutical composition may contain between 0.1% and 100% (w / w) of the first active ingredient.

[0050] "Treatment" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, or preventing the condition or disorder or one or more symptoms of such condition or disorder to which the term applies, unless otherwise indicated. The term "treat" as used herein refers to the act of treating, as "treat" is defined immediately above.

[0051] The "effective amount" described in the present invention refers to an amount sufficient to induce a desired biological response. The effective amount of the active ingredient of the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. The effective amount is the amount of the only active ingredient described in the present invention in a single dose. In certain embodiments, the effective amount is the combined amount of the only active ingredient described in the present invention in multiple doses.

[0052] The "therapeutically effective amount" described in the present invention is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or sufficient to delay or minimize one or more symptoms associated with the disorder. The therapeutically effective amount of Herba Artemisiae Argyi and its extracts means the amount of the therapeutic agent that provides a therapeutic benefit in the treatment of the disorder alone or in combination with other therapies. The term "therapeutically effective amount" may encompass an amount that improves overall therapy, reduces or avoids symptoms, signs or causes of a disorder, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat any of the diseases or disorders described.

[0053] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercial products in the technical field. It is worth noting that the medicinal materials and reagents used in the examples of the present invention are shown in Table 1.

[0054] Table 1 Experimental herbs and reagents

[0055] name factory Golden thread half maple lotus Sichuan Xingzhikang Biotechnology Co., Ltd. (collected in June 2021, origin: Liuzhou, Guangxi) Silybin Shanghai Aladdin Biochemical Technology Co., Ltd. (Batch No.: S109809) <![CDATA[Carbon tetrachloride (CCl 4 )]]> Shanghai MacLean Biochemical Technology Co., Ltd. (CAS: 56-23-3) N-Dimethylnitrosamine (DMN) Shanghai MacLean Biochemical Technology Co., Ltd. (Cat. No. S109809) Corn Oil Yihai Kerry Golden Dragon Fish Cereals, Oils and Foodstuffs Co., Ltd. 4% paraformaldehyde Wuhan Saiweier Biotechnology Co., Ltd. Isoflurane Shenzhen Ruiwode Life Science Co., Ltd. Reagent test kit Nanjing Jiancheng Bioengineering Institute

[0056] Example 1: Alcohol extract of Radix Glehniae

[0057] The preparation method is as follows: according to the material-liquid mass ratio of 1:10, the Herba Artemisiae Argyi and 70% ethanol by volume are mixed, loaded into a reflux device, heated and refluxed for extraction, extracted twice, each time with reflux for 1.5 hours, filtered, combined the filtrate, and concentrated by a rotary evaporator to a drug solution concentration of 1g crude drug / mL.

[0058] [The preventive and therapeutic effects of Jinlu Banfenghe on liver fibrosis]

[0059] The carbon tetrachloride and dimethylnitrosamine (DMN) models were used to comprehensively evaluate the preventive and therapeutic effects of the alcohol extract of Semiliquidambar cathayensis HTChang on liver fibrosis, providing a basis for the clinical drug transformation of Semiliquidambar cathayensis HTChang.

[0060] Example 2 Preventive and therapeutic effects of Radix Glechomae var. chinensis on carbon tetrachloride-induced liver fibrosis

[0061] 1. Experimental Animals

[0062] Sixty SPF-grade male SD rats, 6-8 weeks old, with a body weight of 200±20 g, were provided by the Guangdong Provincial Medical Laboratory Animal Center. The license number for the experimental unit to use is: SYXK(Yue)2022-0125, and the license number is: SYXK(Yue)2022-0002. The animals were housed in the SPF-grade barrier environment of the Experimental Animal Center of Guangdong Pharmaceutical University. All operations during the experiment complied with the experimental animal ethics principles of the Experimental Animal Center of Guangdong Pharmaceutical University.

[0063] 2. Experimental methods

[0064] 2.1 Grouping of experimental animals

[0065] The SD rats were raised under SPF-grade experimental conditions for one week, with free access to food and water. The room temperature was 23±2 °C, the humidity was 50±5%, and the light-dark cycle was controlled at 12 hours. The rats were randomly divided into a normal control group (Con), a model group (Mod), a positive drug group (SFJ), a low-dose group of administering Semiliquidambar cathayensis Hance (LSC), a medium-dose group of administering Semiliquidambar cathayensis Hance (MSC), and a high-dose group of administering Semiliquidambar cathayensis Hance (HSC), with 10 rats in each group. Among them, the medium-dose group was the equivalent dose group after converting the human drug dosage.

[0066] 2.2 Establishment of liver and kidney fibrosis models

[0067] Modeling was carried out from the 1st to the 4th week of the experiment, 3 times a week, by intraperitoneal injection;

[0068] The normal control group was given intraperitoneal injection of corn oil;

[0069] In the remaining groups, a 25% CCl 4 corn oil suspension (V CCl4 :V 玉米油 = 1:3) was intraperitoneally injected at a dose of 2 ml / kg from the 1st to the 2nd week; from the 3rd to the 4th week, a 25% CCl 4 corn oil suspension (VCCl 4 :V 玉米油 = 1:3) was intraperitoneally injected at a dose of 1 ml / kg.

[0070] The total modeling dose was 4.5 ml / kg.

[0071] 2.3 Drug treatment

[0072] From the 3rd to the 4th week of the experiment, gavage administration was carried out once a day, and administration continued for 3 days after the end of modeling in the 4th week; the normal control group and the model group were gavaged with an equal amount of distilled water, and the remaining groups were given the corresponding doses of drugs according to the groups. The positive drug used was silybin. The recommended daily dosage of Semiliquidambar cathayensis Hance for humans is 15 g / 60 kg, and for rats (200 g), the equivalent dose is 1.6 g / kg, which is 6.3 times the human recommended dose.

[0073] Specifically:

[0074] For the positive drug group, the dosage was 100 mg / kg;

[0075] The low-dose Jinlu Banfenghe administration group had a dosage of 0.525 g / kg;

[0076] The medium-dose Jinlu Banfenghe group received a dose of 1.575 g / kg;

[0077] The high-dose Jinlu Banfenghe administration group had a dosage of 4.725 g / kg.

[0078] The specific experimental process involves Figure 1 shown.

[0079] 2.4 General behavioral observations

[0080] The weight changes of rats were recorded (twice a week), and the changes of coat color and mental state of rats were observed.

[0081] 2.5 Sample collection and processing

[0082] After the last administration, rats in each group were fasted (but not water) for 12 hours. Subsequently, isoflurane was used for inhalation anesthesia, and blood was collected through the abdominal aorta. After the blood was allowed to stand for 2 hours, it was centrifuged at 3500rpm and 4°C for 15 minutes to separate the upper serum or plasma and store it in a -80°C refrigerator. After blood collection, the liver, kidney, heart, lung, colon and colon contents were removed. The liver was photographed, weighed, embedded and frozen, the colon contents were also frozen, and the remaining tissues were embedded and frozen.

[0083] 2.6 Statistical analysis

[0084] GraphPad Prism was used for plotting, and one-way analysis of variance was used for comparison of means among multiple groups. P < 0.05 was considered statistically significant, and P < 0.001 was considered highly statistically significant.

[0085] 3. Experimental results

[0086] 3.1 Weight and diet

[0087] like Figure 2As shown in the figure, during the 4-week experimental period, in the initial stage, the average body weight of all groups was basically the same. The weight of rats in the Con group gradually increased, their fur was shiny, their movements were agile, and their mental state was good. No obvious hair loss or diarrhea was observed. Compared with the Con group, the weight of the other groups increased slowly, and a downward trend appeared at the end of the second week of modeling. From the third week, the modeling dose was halved, and drug administration began. The weight increase trend of Mod and each drug administration group (LSC, MSC, HSC and SFJ) increased, and at the end of the experiment, the mean body weight of each drug administration group exceeded that of the Mod group. However, due to the large difference in body weight within the group, no statistical difference could be produced, as shown in Table 2. According to Figure 3 The last weight gain showed that there were significant differences between the Con group and each drug-treated group and the Mod group, indicating that the drug-treated group improved the condition of the rats.

[0088] Table 2 Final weighing data

[0089] Group Weight (g) Con 408.30±33.25 Mod 337.77±21.55 SFJ 347.91±22.56 LSC 343.72±18.34 MSC 345.46±20.31 HSC 347.70±29.47

[0090] 3.2 Liver morphology and index

[0091] Figure 4 The macroscopic morphology of rat liver. It can be observed that the liver surface of rats in the Con group is smooth and normal in color; on the contrary, the liver surface of rats in the Mod group is rough, granular, and dull yellow in color. The liver lesions of rats in each drug-treated group were alleviated to varying degrees, among which the improvements in the HSC group and the SFJ group were the most obvious. This shows that both positive drugs and Herba Artemisiae Radix have significant protective effects on the liver.

[0092] Figure 5 A and B are the liver weight and liver index of rats in each group, respectively. Compared with the Con group, the liver weight and liver index of rats in the Mod group were significantly increased (p < 0.001), which may be caused by the progression of liver fibrosis, hepatocyte swelling or inflammatory response. Compared with the Mod group, the liver index of rats in each drug-treated group was significantly reduced (p < 0.001), close to the level of the Con group. This result shows that the drug-treated group has a certain protective effect on the liver of rats.

[0093] 3.3 Liver function level

[0094] like Figure 6As shown in Figures A and B, during the 4-week experiment, on the second day after the last modeling each week, 6 rats in each group were randomly selected for orbital blood sampling, centrifugation, and serum collection. It can be seen that there was no significant change in the serum liver function level of rats in the Con group, indicating that intraperitoneal injection of corn oil had no effect on the liver; the serum ALT and AST levels of rats in the Mod group increased week by week, and the values ​​reached a bottleneck in the third week. With the increase in the administration time, the ALT and AST levels of each administration group gradually decreased, among which the HSC group had the most significant reduction effect (p < 0.001). The ALT and AST levels of the SFJ group and the LSC group were higher than those in the third week at the fourth week, but were still lower than the levels of the Mod group as a whole, indicating that the administration has a certain protective effect on the liver, but low-dose administration is less effective in combating the continued progression of modeling damage.

[0095] Figure 7 Figures A and B are the ALT and AST levels of rat serum after the experiment. It can be seen that after the modeling was stopped, the liver function of rats in each drug-treated group was significantly restored to a normal level, and there was no obvious gradient difference between the drug-treated groups. However, the liver function of rats in the Mod group showed no obvious self-recovery trend after the modeling was stopped, indicating that the positive drug and Jinlu Banfenghe have a significant liver-protecting effect.

[0096] 3.4 Liver tissue pathology sections

[0097] Figure 8 A in the middle shows the Sirius red staining results of the liver. In the rats of the Con group, the liver tissue structure was basically normal, the hepatic cords were arranged regularly and tightly, no inflammatory cell infiltration and fibrosis changes were observed in the liver parenchyma, and no pseudolobules were found. Compared with the Con group, the liver lobule structure of the rats in the Mod group was disordered, the hepatocytes were fatty degenerated and necrotic, and there was inflammatory cell infiltration. The liver lesions of the rats in each drug-treated group were alleviated to varying degrees, as shown in the significant reduction of pseudolobular lesions, the improvement of liver lobule structure, the significant reduction of fibrous tissue proliferation, and the reduction of inflammatory cell infiltration. Four fields of view were selected for each pathological section, and statistical analysis was performed using ImageJ, as shown in the figure. Figure 8 As shown in B. The results showed that the fibrosis area of ​​each drug-treated group was significantly smaller than that of the Mod group, and the difference was statistically significant (p < 0.001).

[0098] Fig. 9 Figure A shows the Masson staining result of the liver. Similar to the Sirius red staining result, the liver tissue structure of rats in the Con group was normal, while the liver of rats in the Mod group showed significant fibrosis and structural disorder. The degree of fibrosis in rats in each drug-treated group was significantly reduced, and the liver lobule structure was improved. The results of Masson staining were quantitatively analyzed using ImageJ software. Fig. 9As shown in B. It was found that the fibrosis area of ​​each drug-treated group was significantly smaller than that of the Mod group (p < 0.05, p < 0.01 or p < 0.001), which further verified the alleviating effect of Jinlu Banfenghe administration on liver fibrosis.

[0099] Fig.10 Figures A, B, and C show the Ishake scores, percentage of fatty degeneration, and percentage of ballooning degeneration in each group of rats, respectively. Fig.10 A in Figure 2 shows that compared with the Con group, the Ishake score of rats in the Mod group was significantly increased (p < 0.001), indicating that the degree of liver fibrosis was aggravated. Compared with the Mod group, the Ishake score of the SFJ group did not decrease significantly (p > 0.05), while the Ishake scores of rats in the MSC and HSC groups decreased significantly (p < 0.001), indicating that although silybin has a significant liver-protecting effect, it cannot improve the already formed liver fibrosis, and Jinlu Banfenghe has a better improvement effect.

[0100] from Fig.10 As shown in B, in terms of the percentage of fatty degeneration, the percentage of fatty degeneration of hepatocytes in the Mod group was significantly higher than that in the Con group (p < 0.001). The percentage of fatty degeneration in each drug-administered group (SFJ, MSC, and HSC) was significantly reduced (p < 0.001), close to the level of the Con group, indicating that drug administration can effectively reduce fatty degeneration of hepatocytes.

[0101] from Fig.10 As shown in C, the percentage of ballooning in the Mod group was significantly higher than that in the Con group (p < 0.001), indicating more severe liver cell damage. The percentage of ballooning in each drug-treated group was significantly reduced (p < 0.001), indicating that Jinlu Banfenghe has a good protective effect on liver cells.

[0102] In summary, the statistical results of Ishake score, percentage of fatty degeneration and percentage of ballooning degeneration further verified the significant alleviating effect of Radix Glechomae Argyi on liver fibrosis, indicating that Radix Glechomae Argyi has important potential in protecting liver structure and function.

[0103] Example 3: The preventive and therapeutic effects of Radix Glehniae on DMN-induced liver fibrosis

[0104] 1. Experimental Animals

[0105] 60 male Kunming (KM) mice, weighing 20-25 g, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd., animal qualification certificate number: SCXK (Guangdong) 2020-0051. All operations during the experiment complied with the experimental animal ethics principles of the Experimental Animal Center of Guangdong Pharmaceutical University.

[0106] 2. Experimental methods

[0107] 2.1 Experimental Animal Grouping

[0108] KM mice were kept in SPF experimental conditions for one day, with free access to food and water, room temperature of 23±2℃, humidity of 50±5%, and light-dark cycle of 12 hours. Mice were randomly divided into normal control group (Con), model group (Mod), positive drug group (Silibinin, SFJ), low-dose Jinlu Banfenghe administration group (LSC), medium-dose Jinlu Banfenghe administration group (MSC), and high-dose Jinlu Banfenghe administration group (HSC), with 10 mice in each group. The low dose is the equivalent dose group after the human dose is converted.

[0109] 2.2 Establishment of liver fibrosis model

[0110] The model was established in the 1st to 3rd week of the experiment, and the mice were intraperitoneally injected 3 times a week. The Con group was gavaged with normal saline. The other groups were intraperitoneally injected with 0.3% DMN normal saline solution at a dose of 10 mg / kg in the 1st to 3rd week. The injection dosage changed with the body weight measurement results.

[0111] 2.3 Drug treatment

[0112] In the first week of the experiment, the drug was administered by gavage once a day starting from the day before modeling, and continued for one day after modeling in the third week; the Con group and the Mod group were gavaged with the same amount of distilled water, and the other groups were given corresponding doses of drugs according to the group. Silibinin (SFJ) was used as a positive drug. The recommended daily dosage of Jinlu Banfenghe for humans is 15g / 60kg, and the equivalent dose for mice (50g) is 9.1 times the recommended daily dosage for humans, that is, 2.275g / kg.

[0113] Specifically:

[0114] For the positive drug group, the dosage was 50 mg / kg;

[0115] The low-dose Jinlu Banfenghe administration group had a dosage of 2.275 g / kg;

[0116] The medium-dose Jinlu Banfenghe group received a dose of 6.825 g / kg;

[0117] The high-dose Jinlu Banfenghe administration group had a dosage of 13.65 g / kg.

[0118] The specific experimental process involves Fig.11 shown.

[0119] 2.4 General behavioral observations

[0120] The weight changes of mice were recorded (measured every 3 days), and the changes of coat color and mental state of mice were observed.

[0121] 2.5 Sample collection and processing

[0122] After the last administration, mice in each group were fasted (but not water) for 12 hours. Subsequently, isoflurane was used for inhalation anesthesia, and blood was collected from the eyeballs. After the blood was allowed to stand for 2 hours, it was centrifuged at 3500rpm and 4°C for 15 minutes to separate the upper serum or plasma and store it in a -80°C refrigerator. After blood collection, the liver, colon and colon contents were removed. The liver was photographed, weighed, embedded and frozen, the colon was cut into 1-2cm and 4-5cm pieces for embedding, and the colon contents were frozen.

[0123] 2.6 Statistical analysis

[0124] GraphPad Prism was used for plotting, and one-way analysis of variance was used for comparison of means among multiple groups. P < 0.05 was considered statistically significant, and P < 0.001 was considered highly statistically significant.

[0125] 3. Experimental results

[0126] 3.1 Liver morphology

[0127] Fig.12 The macroscopic morphology of mouse liver. It can be observed that the liver surface of mice in the Con group was smooth and normal in color; on the contrary, the liver surface of mice in the Mod group was rough, granular, and dull in color. The liver lesions of mice in each drug-treated group were alleviated to varying degrees, indicating that the positive drugs silybin and golden vine half maple have a protective effect on the liver.

[0128] 3.2 Liver function level

[0129] After the last administration, mice in each group were fasted (but not water) for 12 hours. Then, isoflurane was used for inhalation anesthesia and blood was collected from the eyeball. After the blood was allowed to stand for 2 hours, it was centrifuged at 3500 rpm and 4°C for 15 minutes to collect serum. Then, AST and ALT were tested strictly according to the instructions of the kit. The results are shown in the figure. Fig.13 shown.

[0130] The results show that Fig.13 As shown in Figure A, compared with the control group (Con), the ALT level in the serum of the model group (Mod) mice was increased (p < 0.001); however, compared with the Mod group, the ALT level in the serum of the positive drug group (SFJ) and the drug-treated group (LSC, MSC and HSC) was decreased (p < 0.001), and it was dose-dependent. Fig.13As shown in Figure B, compared with the Con group, the serum AST level in the Mod group increased (p < 0.001); however, compared with the Mod group, the serum AST level in the SFJ group and the HSC group decreased (p < 0.001 or p < 0.05). The above results indicate that different doses of Jinlu Banfenghe can alleviate the liver injury caused by DMN to varying degrees, suggesting that Jinlu Banfenghe has a protective effect on DMN-induced liver injury.

[0131] 3.3 Liver tissue pathology sections

[0132] Masson staining of the liver Fig.14 As shown. The results showed. No inflammatory cell infiltration and collagen fiber proliferation were found in the portal area of ​​mice in the Con group, and only a small amount of collagen fibers were found around blood vessels such as the central vein. A large amount of collagen fiber proliferation was observed in the mice in the Mod group, and collagen fibers were seen extending outward along the portal area or the inflammatory necrosis area. The degree of fibrosis in mice in each drug-treated group (SFJ, LSC, MSC and HSC) was significantly reduced, and the liver lobule structure was improved. The fibrosis area was significantly smaller than that in the Mod group (p < 0.05), which further verified the alleviating effect of Jinlu Banfenghe administration on liver fibrosis.

[0133] In summary, Herba Artemisiae Radix Panacis has a significant liver protective effect and can effectively reduce the progression of liver fibrosis. These results provide experimental basis and scientific support for the potential application of Herba Artemisiae Radix Panacis in the treatment of liver diseases.

[0134] The above is a further description of the present invention in conjunction with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. Use of Radix Angelicae Pubescentis, its extract or its pharmaceutical composition in the preparation of drugs for preventing and treating liver fibrosis.

2. Use of Radix Angelicae Pubescentis, its extract or its pharmaceutical composition in the preparation of a medicament for preventing and treating liver diseases by improving liver fibrosis.

3. Use of Radix Angelicae Pubescentis, its extract or its pharmaceutical composition in the preparation of drugs for preventing and treating liver diseases.

4. The use according to claim 2 or 3, characterized in that: The liver disease includes at least one of non-alcoholic fatty liver disease, alcoholic fatty liver disease, liver cyst, benign tumor, hepatitis and cirrhosis.

5. The use according to any one of claims 1 to 3, characterized in that: The Herba Lycopodiellae extract is a water extract and / or an alcohol extract of Herba Lycopodiellae.

6. The use according to claim 5, characterized in that: The preparation method of the water extract of Jinlu Banfenghe is as follows: adding Jinlu Banfenghe to water for reflux extraction, and separating the solid and liquid to obtain; The preparation method of the alcohol extract of Radix Angelicae Pubescentis comprises the following steps: adding Radix Angelicae Pubescentis to alcohol for reflux extraction, and performing solid-liquid separation to obtain the alcohol extract.

7. The use according to claim 5, characterized in that: The volume fraction of the alcohol is 10-90%, and the amount of the alcohol added is 5-30 times the weight of the golden vine half maple; the extraction is performed at least once, each time for 0.5-3 hours.

8. The use according to any one of claims 1 to 3, characterized in that: The pharmaceutical composition comprises Radix Angelicae Pubescentis or its extract and pharmaceutically acceptable excipients.

9. The use according to claim 1, characterized in that: The pharmaceutical composition is in the form of granules, tablets, capsules, powders, pills, sprays or liquid preparations.

10. A pharmaceutical composition, characterized in that The invention is prepared from Radix Glehniae or its extract and pharmaceutically acceptable auxiliary materials.

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