Application of formononetin

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of formononetin in prevention and treatment of duck hepatitis B virus. According to the invention, the anti-DHBV effect of formononetin is proved for the first time. According to the application disclosed by the invention, the formononetin is proved to have a very good antiviral effect on DHBV by virtue of research of various methods, the action mechanism of the formononetin is clarified, a foundation is laid for developing a novel drug for resisting DHBV and discussing the action mechanism of the drug, and the formononetin has a very good application prospect in the aspect of preventing and treating duck hepatitis B.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to an application of formononetin. Background Art

[0002] Hepatitis B is a disease caused by the hepatitis B virus (HBV) and characterized by liver inflammation and necrosis. The hepatitis B virus is a DNA virus, and its genome is a double-stranded, circular, incompletely closed DNA. The outermost layer of the virus is the outer membrane or envelope, and its inner layer is the core. The nucleoprotein is the core antigen (HBcAg), which cannot be detected in the serum. Under an electron microscope, three types of particles can be seen in the serum of HBsAg-positive people: round and filamentous particles with a diameter of 22nm, and a smaller number of spherical particles with a diameter of 42nm, also known as Dane particles, which are complete HBV particles.

[0003] Duck Hepatitis B virus (DHBV) is the first hepatitis B virus discovered from an avian host. It belongs to the avian hepatotropic DNA virus. All members of this family show similar genome organization and replication strategies in terms of hepatotropism and virion morphology, and cause species infection, resulting in transient (acute) or persistent infection. The virus has a diameter of 40nm, and the DNA genome is circular, partially single-stranded. The endogenous DNA polymerase associated with the virus can convert the genome into a double-stranded circular form of about 3,000 base pairs in size. Analysis of viral DNA in organs of infected birds showed that hepatovirus infection mainly occurs in hepatocytes of the liver, preferentially localized in the liver, which is the site of viral replication and releases infectious virus particles and non-infectious surface antigen particles into the blood.

[0004] Duck hepatitis B virus is a pathogen that can cause duck hepatitis. It is highly contagious and pathogenic. DHBV infection can cause liver damage in ducks, seriously affecting the growth and reproduction of ducks. In addition, the virus is mainly transmitted through blood and body fluids, including blood, feces, oral secretions, etc. It is easy for the virus to spread in densely-stocked or unclean environments, especially in cases of severe liver damage, which can easily cause an outbreak and cause great economic losses to the duck farming industry.

[0005] Currently, there is no effective antiviral drug against DHBV in clinical practice. Immunization of laying ducks or ducklings with attenuated vaccines is the main way to control the disease. Western medicines such as amantadine, rimantadine, acyclovir, morpholine (bi)guanidine (Viralin), ribavirin, and their salts and esters, single and compound preparations are prohibited. Therefore, in the context of the urgent search for safer and more effective therapeutic drugs, Chinese herbal medicines have received widespread attention due to their wide sources, high safety, and biodegradability. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an application of formononetin, especially in the prevention and treatment of duck hepatitis B virus.

[0007] Formononetin (7-hydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one, Formononetin, referred to as FOR), has a molecular formula of C 16 H12O4, molecular weight 268.26408, extract of the inflorescence and flowering branches and leaves of the legume plant Trifolium purpurogenum. It is mainly found in legumes such as the root of Astragalus, the inflorescence and flowering branches and leaves of Trifolium purpurogenum, Pueraria lobata and Millettia reticulata. It belongs to isoflavone compounds and is the representative component of isoflavone compounds in Astragalus. Formononetin is a white powder, which is easily soluble in methanol, ethyl acetate, ether and dilute alkaline solution. Formononetin has multiple biological activities such as anti-inflammatory, antioxidant, anti-apoptosis, lipid regulation, anti-thrombosis and tumor inhibition. It can be used in the treatment of cardiovascular and cerebrovascular diseases, tumors, diabetes and its complications and other diseases, and has a wide range of clinical application potential. However, there has been no report on the anti-DHBV effect of formononetin.

[0008] The present invention also provides an application of formononetin in preparing a medicine for preventing and / or treating duck hepatitis B virus.

[0009] The present invention uses a Chinese medicine monomer compound, formononetin, which refers to a chemical substance with a certain molecular weight, molecular formula, physicochemical constants and a determined chemical structure obtained by extraction, separation and purification from Chinese medicine. The Chinese medicine monomer compound has simple quality control, eliminates the interference of a large number of unnecessary components and other possible restrictive, inhibitory and antagonistic components coexisting in Chinese medicine, makes the effect more specific, and is conducive to the study of pharmacodynamics and mechanism. The present invention has found through research that formononetin has a good inhibitory effect on DHBV, and has a good application prospect in the prevention and treatment of duck hepatitis B.

[0010] In a specific embodiment provided by the present invention, the formononetin prevents and / or treats duck hepatitis B virus by reducing the level of duck hepatitis B surface antigen and / or inhibiting the expression of duck hepatitis B e antigen.

[0011] In a specific embodiment provided by the present invention, the formononetin prevents and / or treats duck hepatitis B virus by reducing one or more of the following index levels:

[0012] (1) Alanine aminotransferase;

[0013] (2) Asparagine aminotransferase;

[0014] (3) alkaline phosphatase;

[0015] (4)Total bilirubin.

[0016] In a specific embodiment provided by the present invention, the formononetin prevents and / or treats duck hepatitis B virus by inhibiting duck interleukin 6 and / or duck interleukin 1β.

[0017] In a specific embodiment provided by the present invention, the formononetin prevents and / or treats duck hepatitis B virus by improving the total antioxidant capacity of duck liver tissue.

[0018] In a specific embodiment provided by the present invention, the formononetin prevents and / or treats duck hepatitis B virus by inhibiting hepatocyte fibrosis.

[0019] The present invention establishes a duck hepatitis B animal model, evaluates the efficacy of formononetin in resisting duck hepatitis B virus and protecting the liver after oral administration, and uses acyclovir as a positive control drug, and uses fluorescent quantitative PCR method, Masson staining and other technologies to prove through experiments at various levels that formononetin has an antiviral effect on DHBV, proving that formononetin has an anti-DHBV effect in vitro, is expected to become a new drug for preventing and treating duck hepatitis B, and has a good application prospect in the prevention and treatment of duck hepatitis B drugs. It can be seen that the present invention conducts experimental research on formononetin against DHBV, and provides valuable theoretical basis and reference for the clinical use of DHBV.

[0020] The present invention also provides an application of formononetin in preparing a medicine for preventing and / or treating duck hepatitis B.

[0021] In a specific embodiment provided by the present invention, the duck hepatitis B is caused by duck hepatitis virus.

[0022] In a specific embodiment provided by the present invention, the effective dose of formononetin for preventing and / or treating duck hepatitis B is 50-200 mg / kg; specifically, it can be 50 mg / kg, 100 mg / kg, 200 mg / kg or a range between any two of the above values.

[0023] In a specific embodiment provided by the present invention, the drug further comprises a pharmaceutically acceptable carrier. By adding different carriers, the drug can be prepared into different dosage forms, including but not limited to injections, oral preparations, sustained-release preparations, and the like.

[0024] In a specific embodiment provided by the present invention, the medicine also includes one or more of other traditional Chinese medicines, vaccines and biological preparations; that is, the formononetin can be used simultaneously with one or more of other traditional Chinese medicines, vaccines and biological preparations.

[0025] In a specific embodiment provided by the present invention, the formononetin can be used in combination with one or more of Patrinia scabra, Portulaca oleracea, Tripterygium wilfordii, Lobelia chinensis, Gentiana scabra and the like.

[0026] Duck hepatitis B virus and human hepatitis B virus belong to the same family of hepadnaviridae, and the genome, replication mode and pathogenesis of duck hepatitis B virus are similar to those of human hepatitis B virus. Therefore, the duck hepatitis B model has become a conventional model for screening and evaluating anti-HBV drugs in recent years. Based on this, it can be inferred that the formononetin also has the effect of preventing and treating human hepatitis B virus.

[0027] Therefore, the present invention also provides a use of formononetin in preparing a medicine for preventing and / or treating hepatitis B virus.

[0028] Compared with the prior art, the present invention proves for the first time the anti-DHBV effect of formononetin. The present invention verifies through multiple methods that formononetin has a good antiviral effect on DHBV, and clarifies its mechanism of action, laying a foundation for the development of new anti-DHBV drugs and exploring the drug mechanism of action, and has a good application prospect in the prevention and treatment of duck hepatitis B.

[0029] Moreover, formononetin has the following advantages: anti-tumor, lipid-lowering, anti-arrhythmic, antibacterial, antispasmodic, anti-lipid peroxidation, estrogen and other effects, can improve atherosclerotic lesions, and also has the effects of scavenging oxygen free radicals and inhibiting the proliferation of vascular smooth muscle cells. In addition, the effective parts of traditional Chinese medicine or monomer compounds of traditional Chinese medicine are simple to control in quality, and the interference of a large number of unnecessary components and other possible restrictive, inhibitory and antagonistic components coexisting in traditional Chinese medicine is eliminated, making the effect more specific, which is conducive to the study of pharmacodynamics and mechanism. Moreover, it has been verified that formononetin has a significant anti-DHBV effect within a certain dose.

[0030] The invention uses a duck to establish an animal model of hepatitis B, explores the anti-duck hepatitis B virus effect of formononetin for the first time, and provides a theoretical basis for developing a new drug for treating viral duck hepatitis B. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Agarose gel electrophoresis diagram of screening serum DHBV DNA positive ducks;

[0032] Figure 2 It is the inhibition rate curve of formononetin on DHBV;

[0033] Figure 3 This is the result diagram of the effect of formononetin on DHBsAg;

[0034] Figure 4 This is the result diagram of the effect of formononetin on DHBeAg;

[0035] Figure 5 This is the result diagram of the effect of formononetin on ALT;

[0036] Figure 6 This is the result diagram of the effect of formononetin on AST;

[0037] Figure 7 This is the result diagram of the effect of formononetin on ALP;

[0038] Figure 8 This is the result diagram of the effect of formononetin on TP;

[0039] Fig. 9 This is the result diagram of the effect of formononetin on ALB;

[0040] Fig.10 This is the result diagram of the effect of formononetin on GGT;

[0041] Fig.11 This is the result diagram of the effect of formononetin on TBIL;

[0042] Fig.12 This is the result diagram of the effect of formononetin on IL-6;

[0043] Fig.13 This is the result diagram of the effect of formononetin on IL-1β;

[0044] Fig.14 This is the result diagram of the effect of formononetin on MDA;

[0045] Fig.15 This is the result diagram of the effect of formononetin on SOD;

[0046] Fig.16 This is the result diagram of the effect of formononetin on GSH-PX;

[0047] Fig.17 This is the result diagram of the effect of formononetin on CAT;

[0048] Fig.18This is the result diagram of the effect of formononetin on T-AOC;

[0049] Fig.19 This is the result of the effect of formononetin on duck liver pathological sections (Masson staining);

[0050] Fig. 20 This is the effect of formononetin on duck liver pathological sections (HE staining) results;

[0051] Fig.21 This is the result diagram of the effect of formononetin on Ishak Score;

[0052] In the above figures, compared with the acyclovir group, **P<0.01, *P<0.05; compared with the model group, aa P<0.01, a P<0.05. DETAILED DESCRIPTION

[0053] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0054] Statistical analysis of the following examples of the present invention: All experiments were repeated at least 3 times independently, and one-way analysis of variance was performed using SPSS 17.0 software. All data were expressed as Mean ± SD, and Student's t test was used for statistical analysis.

[0055] The duck hepatitis B virus used in the following examples of the present invention was isolated and donated by the Poultry Research Institute of Shandong Academy of Agricultural Sciences. At present, there is no clear genotype system classification method for duck hepatitis B virus like human HBV. The academic community mostly classifies and describes DHBV based on the type of isolated host, geographical distribution and gene sequence variation characteristics. DHBV is mainly manifested in differences in several evolutionary branches and strain lineages, rather than a strict "genotype" division. In other words, existing studies are more about distinguishing DHBV strains by place of origin and host species, and determining their genetic relationships through molecular evolution analysis.

[0056] Experimental Example 1 Amplification and Preparation of Duck Hepatitis B Virus

[0057] 50 SPF duck embryos were incubated for about 28 days. When the ducklings hatched, they were adaptively raised until 3 days old and injected with duck hepatitis B virus through the jugular vein at 100 μL / duck.

[0058] Experimental Example 2 Establishment of duck hepatitis B animal model

[0059] SPF duck embryos were incubated for about 28 days, and when the ducklings were hatched, they were adaptively raised until 3 days old. 50 ducklings were injected with duck hepatitis B virus through the tibial vein, 100 μL / duck, and the remaining 7 were fed normally. On the 13th day (T0), 1 mL of blood was collected from the jugular vein of the ducks and placed in a 1.5 mL centrifuge tube with the corresponding number. The tube was centrifuged at 4000 rpm / min for 10 min at room temperature for 2 hours, and the supernatant was taken and stored for later use. 35 DHBV-DNA-positive ducks were screened by PCR (primer sequence DHBV-S shown in Table 1) and agarose electrophoresis to establish a duck hepatitis B animal model.

[0060] like Figure 1 As shown, the electrophoresis detection result of the amplified product showed a single bright band, which was consistent with the expected fragment size. The sample serum was judged to be positive, with a positive rate of 85%, which met expectations, and the duck hepatitis B model was successfully established.

[0061] Experimental Example 3 Animal Grouping and Dosing

[0062] Thirty-five SPF ducks with positive DHBV-DNA were randomly divided into five groups, with seven ducks in each group: high-, medium-, and low-dose groups of formononetin (FOR H Group, FOR M Group, FOR L The ducks were divided into three groups (groups) (200 mg / kg, 100 mg / kg, and 50 mg / kg, respectively), acyclovir positive control group (PC group) (200 mg / kg), and model control group (Mod group). The drugs were administered by gavage once a day for 14 consecutive days. The model control group was given 2 mL of distilled water per day, and the acyclovir group was given 200 mg / kg of acyclovir solution per day.

[0063] Before administration (T0), 7 days after administration (T7), 14 days after administration (T 14 ) and 5 days after drug withdrawal (P5), 5 ducks were randomly selected from each group, 1.5 mL of blood was collected from the right jugular vein of the ducks, and the blood was kept at room temperature for 1 hour. The serum samples were separated and stored in a -80℃ refrigerator for testing. 5 days after drug withdrawal, the ducks were killed and the livers were removed by laparotomy.

[0064] All ducks were housed in different isolators under the same experimental conditions.

[0065] Test Example 4D HBV-DNA virus inhibition rate detection

[0066] The detection of DHBV-DNA in serum was performed by real-time fluorescence quantitative PCR, and the corresponding inhibition rate was calculated using the CT value and the standard curve. The primer sequences are shown in Table 1. The TaKaRa TB Premix Ex Taq TMII (TliRNaseHPlus) kit. Prepare the premix according to the number of samples, mix well, and dispense into 8 tubes, 18 μL each, add 2 μL of sample nucleic acid each, and mix well to obtain the DNA extract. Use real-time fluorescence quantitative PCR instrument to detect and calculate the corresponding inhibition rate. Virus inhibition rate (%) = [(before administration - after administration) / before administration] × 100%. See the results for details. Figure 2 .

[0067] The results showed that after medication, the dynamic curves of virus inhibition rates in the high and low dose groups of formononetin showed a gradual upward trend, while the low dose of acyclovir showed a gradual downward trend. Compared with the acyclovir group, the high dose group had a more significant virus inhibition effect.

[0068] Table 1 qPCR sequences

[0069]

[0070] Experimental Example 5 Effect of Formononetin on Duck Hepatitis B Surface Antigen (DHBsAg) and Duck Hepatitis B e Antigen (DHBeAg)

[0071] The OD value of DHBsAg and DHBeAg in serum was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Jiangsu ELISA Industrial Co., Ltd.), which was purchased from Jiangsu ELISA Industrial Co., Ltd. Strictly follow the instructions for use of the kit, accurately add 50 μL to each well of the enzyme-labeled plate, seal the plate with a sealing film, and incubate at 37°C for 30 minutes. Tear off the sealing film, discard the liquid in the well, and wash each well 5 times with the special buffer of the kit. Subsequently, add 50 μL of enzyme-labeled reagent to each well (except the blank well) and incubate at 37°C for 30 minutes. Repeat the washing of each well 5 times, add 100 μL of color developer, color at 37°C in the dark for 10 minutes, add 50 μL of stop solution, and terminate the reaction. Use a light absorption full-wavelength microplate reader (Shanghai Shanpu Biotechnology Co., Ltd., SP-Max 2300A2) with a wavelength of 450nm to calculate the OD value.

[0072] (1) Effect of formononetin on DHBsAg

[0073] Figure 3 The results of the effect of formononetin on DHBsAg showed that the levels of DHBsAg in the high, medium and low dose groups were significantly higher than those in the T 14 There was a significant decrease at P5, and a slight increase at P5. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01).

[0074] Effect of Formononetin on DHBeAg

[0075] Figure 4The figure shows the effect of formononetin on DHBeAg. The results show that the high, medium and low dose groups all inhibited the expression of DHBeAg in serum. Compared with the acyclovir group and the model group, the expression of DHBeAg in serum was inhibited in T 14 The results were significantly different (P<0.05, P<0.01), and the inhibitory effect became more obvious with the increase of dose, but it rebounded rapidly at P5.

[0076] Experimental Example 6 Effect of Formononetin on Blood Biochemistry

[0077] A fully automatic biochemical analyzer (Hitachi, Japan, 7180 fully automatic blood biochemical analyzer) was used to detect serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), γ-glutamyl transferase (GGT) and total bilirubin (TBIL) levels.

[0078] (1) Effect of formononetin on ALT

[0079] Figure 5 The figure shows the effect of formononetin on ALT. The results show that the ALT levels in the serum of ducks in the high, medium and low dose groups were all reduced after medication, and the inhibitory effect became more obvious with the increase of dose. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01).

[0080] (2) Effect of formononetin on AST

[0081] Figure 6 The figure shows the effect of formononetin on AST. The results show that after administration, the high and low dose groups showed a continuous downward trend, and compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01). At P5, it rebounded, and the high dose group had a smaller rebound, and the effect of inhibiting AST was better. Compared with the acyclovir group and the model group, there was no significant difference (P>0.05); the AST level in the medium dose group decreased and tended to be stable after administration, and compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01).

[0082] (3) Effect of formononetin on ALP

[0083] Figure 7 The results of the effect of formononetin on ALP are shown in the figure. The results show that after medication, the high, medium and low dose groups all showed a decrease, among which the low dose group did not rebound at P5, and the high dose group rebounded slightly. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01). This shows that formononetin can effectively inhibit the activity of ALP in serum.

[0084] (4) Effect of formononetin on TP

[0085] Figure 8 The results of the effect of formononetin on TP showed that the high-dose group showed a trend of continuous increase after medication, while the medium-dose group showed a trend of continuous increase in T 14 There was a short decrease in the low-dose group at T7, and then it rebounded and increased. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01), indicating that formononetin could not effectively inhibit the level of TP in serum.

[0086] (5) Effect of formononetin on ALB

[0087] Fig. 9 The figure shows the effect of formononetin on ALB. The results show that after administration, the high and medium dose groups showed a decrease, but rebounded rapidly at P5, and the results were significantly different from the acyclovir group and the model group (P<0.05, P<0.01); after administration, the low dose group did not show the effect of inhibiting ALB in serum at each period compared with T0, and the results were significantly different from the acyclovir group and the model group (P<0.05, P<0.01).

[0088] (6) Effect of formononetin on GGT

[0089] Fig.10 The figure shows the effect of formononetin on GGT. The results show that the serum GGT levels in the high, medium and low dose groups continued to increase after medication, and the results were significantly different from those in the acyclovir group and the model group (P<0.05, P<0.01). This shows that formononetin cannot effectively inhibit the level of GGT in serum.

[0090] (7) Effect of formononetin on TBIL

[0091] Fig.11 The results of the effect of formononetin on TBIL were shown in Figure 2. The results showed that after administration, the TBIL levels in the serum of the high- and medium-dose groups continued to decrease, while the low-dose group showed a decrease at T7 and continued at T 14 The level of leukemia tended to be stable, but rebounded sharply at P5. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01).

[0092] Experimental Example 7 Effect of Formononetin on Duck Interleukin-6 (IL-6) and Duck Interleukin-1β (IL-1β)

[0093] The OD values ​​of IL-6 and IL-1β in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu ELISA Industrial Co., Ltd.) purchased from Jiangsu ELISA Industrial Co., Ltd. in strict accordance with the instructions of the kit.

[0094] (1) Effect of formononetin on IL-6

[0095] Fig.12 The figure shows the effect of formononetin on IL-6. The results show that the level of duck interleukin-6 in the serum of ducks in the high, medium and low dose groups decreased after medication, and the effect of the high dose group was significant. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01). It rebounded at P5, but still showed an inhibitory effect compared with T0.

[0096] (2) Effect of formononetin on IL-1β

[0097] Fig.13 The figure shows the effect of formononetin on IL-1β. The results show that the IL-1β level in the high-dose group showed a trend of first decreasing and then increasing after administration, but compared with the acyclovir group and the model group, it still had the effect of inhibiting IL-1β, and the results were different (P<0.05, P<0.01).

[0098] Experimental Example 8 Effect of Formononetin on Antioxidant Capacity

[0099] Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (T-AOC) kits were used. About 0.1 g of liver tissue was taken, accurately weighed and placed in a homogenizer filled with physiological saline, and the levels of MDA (nmol / g fresh weight), SOD (U / g fresh weight), GSH-Px (nmol / min / mL), CAT (μmol / min / ML) and T-AOC (μmolTrolox / g fresh weight) in serum were detected according to the instructions of the kits.

[0100] The results are as follows Figure 14 to Figure 18 As shown, Fig.14 for Fig.14 This is the result diagram of the effect of formononetin on MDA. Fig.15 This is the result diagram of the effect of formononetin on SOD. Fig.16 This is the result diagram of the effect of formononetin on GSH-PX. Fig.17 This is the result diagram of the effect of formononetin on CAT. Fig.18 The figure shows the effect of formononetin on T-AOC. Figure 14 to Figure 18It can be seen that after 14 days of oral administration and then 5 days of drug withdrawal, the high-dose group of formononetin can significantly reduce the MDA level in duck liver tissue, increase the GSH-PX and CAT contents in duck liver tissue, and improve the T-AOC level in duck liver tissue. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01); the GSH-PX and CAT contents in duck liver tissue in the medium and low-dose groups increased, but the SOD and T-AOC contents in duck liver tissue were not effectively increased. Compared with the acyclovir group and the model group, the results were significantly different (P<0.05, P<0.01).

[0101] Experimental Example 9 Effect of Formononetin on Histopathology of Duck Liver

[0102] (1) Effects of formononetin on duck liver tissue sections

[0103] 1cm×1cm×1cm liver tissue was taken from the middle of the right lobe of duck liver, fixed with 10% formalin solution, and observed under a light microscope for pathological histological morphology after Masson staining.

[0104] Liver pathological observations show that collagen fibers, mucus, and cartilage are blue, cytoplasm, muscle, cellulose, and red blood cells are red, and nuclei are blue-black. It is mainly used to observe the detection of fibrotic lesions in samples, such as connective tissue hyperplasia and fibrosis. Collagen fibers are blue and are mainly concentrated around blood vessels.

[0105] The results are as follows Fig.19 As shown in the figure, the model group showed obvious portal fibrosis in duck liver cells, and the liver lesions were serious. The fibrosis degree of the high-dose group of formononetin was mild, significantly lower than that of the model group; the medium-dose group showed a certain degree of fibrosis, but also lower than that of the model group; the fibrosis of the low-dose group was more serious, mainly concentrated in some areas, and did not spread on a large scale. This shows that duck liver cells in the high, medium, and low-dose groups of formononetin had different degrees of fibrosis, among which the high-dose group had the best effect in inhibiting liver cell fibrosis.

[0106] (2) Effects of formononetin on pathological changes of duck liver tissue

[0107] Using hematoxylin-eosin (HE) staining and referring to my country's "Program for the Prevention and Treatment of Viral Hepatitis" and the internationally commonly used METAVIR, Ishak and Scheuer scoring systems, the estimation of the lesion range of duck liver tissue specimens is mainly divided into four levels: <1> No lesions; <2> Mild lesions (i.e., the lesions occupy ≤ 1 / 3 of the entire visual field); <3> Moderate lesions (1 / 3<lesions occupying the entire visual field ≤2 / 3); <4> Severe lesions (i.e., lesions occupying more than 2 / 3 of the entire visual field). The distribution of the degree of liver lesions in each group of ducks was counted, and the collated data were statistically analyzed.

[0108] The pathological changes were found to be of four types: fragmentary necrosis around the portal area and the interface around the fibrous septum, confluent necrosis, focal (punctate) lytic necrosis, and portal inflammation. Figure 20-21 As shown in the figure, the range of pathological changes in the duck liver tissues of the model group, the high-, medium-, and low-dose groups of formononetin, and the acyclovir group were estimated and divided into four levels of pathological changes. Compared with the model group, the degree of pathological changes in the duck liver tissues of the acyclovir group and the medium- and high-dose groups was milder (P<0.01), and compared with the acyclovir group, the degree of pathological changes in the duck liver tissues of the high-dose group was milder (P<0.01).

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of formononetin in the preparation of a drug for preventing and / or treating duck hepatitis B virus.

2. The use according to claim 1, characterized in that: The formononetin prevents and / or treats duck hepatitis B virus by reducing the level of duck hepatitis B surface antigen and / or inhibiting the expression of duck hepatitis B e antigen.

3. The use according to claim 1, characterized in that: The formononetin prevents and / or treats duck hepatitis B virus by reducing one or more of the following index levels: (1) Alanine aminotransferase; (2) Asparagine aminotransferase; (3) alkaline phosphatase; (4)Total bilirubin.

4. The use according to claim 1, characterized in that: The formononetin prevents and / or treats duck hepatitis B virus by inhibiting duck interleukin 6 and / or duck interleukin 1β.

5. The use according to claim 1, characterized in that: The formononetin prevents and / or treats duck hepatitis B virus by improving the total antioxidant capacity of duck liver tissue.

6. The use according to claim 1, characterized in that: The formononetin prevents and / or treats duck hepatitis B virus by inhibiting liver cell fibrosis.

7. Use of formononetin in the preparation of a drug for preventing and / or treating duck hepatitis B.

8. The use according to claim 7, characterized in that: The duck hepatitis B is caused by duck hepatitis virus.

9. The use according to any one of claims 1 to 8, characterized in that: The drug also includes a pharmaceutically acceptable carrier; And / or, the drug also includes one or more of Patrinia scabra, Portulaca oleracea, Tripterygium wilfordii, Lobelia chinensis and Gentiana scabra.

10. Use of formononetin in the preparation of a drug for preventing and / or treating hepatitis B virus.

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