Traditional Chinese medicine preparation with liver protection effect as well as preparation method and application of traditional Chinese medicine preparation

Abstract

The invention discloses a traditional Chinese medicine preparation with a liver protection effect and a preparation method and application thereof, and belongs to the technical field of application of medicine extracts. The traditional Chinese medicine preparation is prepared from a gallnut extract, an auxiliary material and an ethanol solution, the mass ratio of the gallnut extract to the auxiliary material is 1: (1-2), and the ratio of the total mass of the gallnut extract and the auxiliary material to the mass volume of the ethanol solution is 100g: (2-5) mL. The traditional Chinese medicine preparation disclosed by the invention has the advantages that the antioxidant capacity of mice is improved, the anti-inflammatory capacity of mice is enhanced, the liver injury of the mice caused by toxicants such as acetaminophen and carbon tetrachloride is relieved, and the intestinal flora structure and liver metabolism condition of the mice are improved; when the compound is used for preparing the liver protection preparation, an antioxidant signal channel of a body can be activated, and oxidation and fibrosis damage of the liver can be relieved, so that the liver function of the body is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of application of drug extracts, and specifically relates to a traditional Chinese medicine preparation with liver-protecting effect, a preparation method and application thereof. Background Art

[0002] Liver fibrosis is a complex pathological process, which is mainly manifested as excessive proliferation of fibrous connective tissue in the liver. It is a pathological repair response to long-term and repeated liver damage, and is usually closely related to factors such as liver inflammation, liver cell damage and hepatic stellate cell (HSC) activation. If not controlled, liver fibrosis may progress to cirrhosis or even liver cancer. At present, in the treatment of liver fibrosis, drugs are mainly used to delay its pathological process in order to control the continued deterioration of the disease. Traditional Western medicine has intervened in the process of liver fibrosis to a certain extent, such as inhibiting collagen synthesis, promoting collagen degradation, and regulating immune responses. However, long-term use of these drugs may bring some side effects, such as drug tolerance, economic burden and potential drug interactions.

[0003] In contrast, traditional Chinese medicine has shown unique characteristics and advantages in the treatment of liver fibrosis. Traditional Chinese medicine usually contains a variety of active ingredients, which can act on the liver fibrosis process through multiple targets and multiple pathways to play a comprehensive conditioning role. In addition, traditional Chinese medicine has relatively few side effects, and its resources are abundant and the price is relatively low, which makes traditional Chinese medicine more advantageous in long-term treatment.

[0004] Therefore, in the treatment of liver fibrosis, it is particularly important to develop a traditional Chinese medicine preparation with low drug resistance, high safety and significant efficacy. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention discloses a Chinese medicine preparation with liver protection effect, a preparation method and application thereof. The Chinese medicine preparation provided by the present invention can activate the Nrf2 (nuclear factor erythroid 2-related factor 2) / HO-1 (heme oxygenase-1) signaling pathway, improve the antioxidant capacity of liver cells, reduce liver damage caused by fibrosis, promote liver function recovery, and thus reduce the impact of chronic liver disease.

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

[0007] The invention discloses a traditional Chinese medicine preparation with liver protection function. The traditional Chinese medicine preparation is prepared from a gallnut extract, an auxiliary material and an ethanol solution; the mass ratio of the gallnut extract to the auxiliary material is 1:(1-2), and the mass volume ratio of the total mass of the gallnut extract and the auxiliary material to the ethanol solution is 100g:(2-5)mL.

[0008] Preferably, the auxiliary materials are β-cyclodextrin and β-mannitol, and the mass ratio of β-cyclodextrin to β-mannitol is 1:(1-4).

[0009] Preferably, the mass ratio of the gallnut extract to the auxiliary material is 1:1.5; the mass volume ratio of the total mass of the gallnut extract and the auxiliary material to the ethanol solution is 100g:2.5mL; and the mass ratio of β-cyclodextrin to β-mannitol is 1:2.

[0010] The present invention also provides a method for preparing the above-mentioned Chinese medicine preparation having liver-protecting effect, comprising the following steps:

[0011] (1) After sieving the Chinese gallnut to remove impurities, crush it into particles with a crusher, take an appropriate amount of the Chinese gallnut particles and place them in a round-bottom flask, then add n-hexane to the round-bottom flask, and after ultrasonic treatment for 1 hour, separate the residue and the extract, repeat the above extraction process for a total of 3 times, and then combine the extracts together;

[0012] (2) using a rotary evaporator to concentrate the combined extracts from step (1) into a thick liquid, and then freeze-drying the thick liquid to obtain a gallnut extract, and storing it at 4° C. for later use;

[0013] (3) The Chinese nut extract of step (2) is uniformly mixed with the above-mentioned auxiliary materials, and ethanol solution is sprayed and continuously stirred until the material reaches a state of "forming a ball when held and falling apart when lightly touched", and then manually granulated using a 14-mesh sieve, and the granules are dried at 60° C., and then sized using a 10-mesh sieve to obtain dark brown granules, thereby obtaining a traditional Chinese medicine preparation.

[0014] Preferably, in step (1), the mass volume ratio of the gallnut particles to n-hexane is 1 g: (3-10) mL.

[0015] Preferably, in step (2), the process parameters of the rotary evaporation are: water bath temperature of 50-60°C, vacuum degree of 100-300 mbar, and rotation speed of 100-120 rpm; the freezing process is: pre-freezing at -40°C for 3-4 hours, then transferring the sample to a freeze dryer, setting the cold trap temperature to -60°C, the tray temperature to 30°C, and freeze-drying for 24 hours.

[0016] Preferably, in step (3), the mass volume fraction of the ethanol solution is 30%.

[0017] The invention also discloses the application of the traditional Chinese medicine preparation in preparing a pharmaceutical product with liver protection effect.

[0018] Preferably, the pharmaceutical product having a liver-protecting effect is a pharmaceutical product for alleviating or treating acute liver injury or liver fibrosis.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The main component of the Chinese medicine preparation disclosed in the present invention is the extract of Galla chinensis, which significantly improves the antioxidant properties in animal serum, including improving the total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, while reducing the content of malondialdehyde (MDA), which enzymes and substances play a key role in protecting cells from free radical damage. In addition, the extract also enhances the anti-inflammatory response and helps to alleviate the inflammatory state by regulating the levels of inflammatory mediators such as interleukin 1β, interleukin 6, interleukin 10 and tumor necrosis factor α (TNF-α). In addition, the extract reduces the deposition of collagen fibers in the liver, thereby alleviating liver damage caused by fibrosis.

[0021] (2) The gallnut extract provided by the present invention can change the types and number of microorganisms in the intestine, promote the growth of beneficial bacteria, inhibit the development of harmful bacteria, help to restore and maintain the integrity of the intestinal barrier function, and reduce the chance of harmful bacteria and their metabolites entering the blood circulation; the intestinal-liver axis connects the intestine and the liver, and changes in the intestinal flora can directly affect the function and metabolic process of the liver, thereby reducing the possibility of these toxic substances directly reaching the liver through the portal vein system and causing damage. In addition, healthy intestinal flora can also generate short-chain fatty acids (SCFAs) such as acetic acid, propionic acid and butyric acid by fermenting indigestible food components. These short-chain fatty acids can be used by the liver as an energy source and have anti-inflammatory and antioxidant properties, further supporting the function of liver cells and promoting their repair and regeneration ability. Therefore, the extract prepared by the present invention indirectly enhances the liver's detoxification ability and metabolic efficiency by adjusting the intestinal flora, and effectively alleviates acute liver damage caused by toxic substances.

[0022] In summary, the traditional Chinese medicine preparation prepared by the present invention can not only inhibit the deposition of collagen fibers in the liver and reduce the risk of liver fibrosis, but also significantly alleviate acute liver damage caused by toxic substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A flow chart of the preparation of the Chinese medicine preparation provided by the present invention and its efficacy detection;

[0024] Figure 2 The effect of the Chinese medicine preparation provided by the present invention on serum interleukin, transaminase and antioxidant enzymes in mice with acute liver injury caused by acetaminophen (CW, MW and OW groups are control group, model group and test group respectively);

[0025] Figure 3This is an image of the effect of the Chinese medicine preparation provided by the present invention on the intestinal flora of mice with acute liver injury caused by acetaminophen (the CW, MW, and OW groups are the control group, model group, and test group, respectively);

[0026] Figure 4 The Chinese medicine preparation provided by the present invention is used for CCl 4 Staining microscopic examination of the improvement of liver fibrosis in mice (CTRL, CCl4, and CCl4+RM groups are the control group, model group, and experimental group, respectively);

[0027] Figure 5 The Chinese medicine preparation provided by the present invention is effective for CCl 4 Effects of serum interleukins, transaminases and antioxidant enzymes on liver fibrosis in mice (CTRL, CCl4, and CCl4+RM groups were control, model, and experimental groups, respectively);

[0028] Figure 6 Fluorescence quantitative graph of the activation of Nrf2 / HO-1 signaling pathway in mouse liver by the traditional Chinese medicine preparation provided by the present invention (CTRL, CCl4, and CCl4+RM groups are the control group, model group, and test group, respectively);

[0029] Figure 7 The immunoblotting and quantitative graph (CTRL, CCl) of the Chinese medicine preparation provided by the present invention on the activation of the Nrf2 / HO-1 signaling pathway in mouse liver 4 , CCl 4 +RM groups were control group, model group, and experimental group respectively);

[0030] Figure 8 The Chinese medicine preparation provided by the present invention is used for CCl 4 Effect diagram of non-target metabolomics in liver of mice with liver fibrosis caused by 4 , CCl 4 +RM groups were control group, model group, and experimental group respectively);

[0031] Fig. 9 The Chinese medicine preparation provided by the present invention is effective for CCl 4 Effect of key strains of intestinal flora on liver of mice with liver fibrosis (CTRL, CCl 4 , CCl 4 +RM groups were control group, model group, and experimental group. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in 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 creative work are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] The invention provides a traditional Chinese medicine preparation with liver protection function. The traditional Chinese medicine preparation is prepared from a gallnut extract, an auxiliary material and an ethanol solution; the mass ratio of the gallnut extract to the auxiliary material is 1:(1-2), and the mass volume ratio of the total mass of the gallnut extract and the auxiliary material to the ethanol solution is 100g:(2-5)mL; the auxiliary materials are beta-cyclodextrin and beta-mannitol, and the mass ratio of beta-cyclodextrin to beta-mannitol is 1:(1-4).

[0036] Among them, the best is that the mass ratio of the above-mentioned gallnut extract to the auxiliary materials is 1:1.5, the mass volume ratio of the total mass of the gallnut extract and the auxiliary materials to the ethanol solution is 100g:2.5mL, and the mass ratio of β-cyclodextrin to β-mannitol is 1:2.

[0037] The present invention also provides a method for preparing the above-mentioned Chinese medicine preparation having liver-protecting effect, and the specific steps are as follows:

[0038] (1) After sieving the Chinese gallnut to remove impurities, the Chinese gallnut was crushed into particles using a crusher. An appropriate amount of Chinese gallnut particles was weighed and placed in a round-bottom flask according to the mass volume ratio of Chinese gallnut particles to n-hexane of 1 g: (3-10) mL. N-hexane was added to the round-bottom flask. After ultrasonic treatment for 1 h, the residue and the extract were separated. The above extraction process was repeated for a total of 3 times, and the extracts were then combined together.

[0039] (2) The parameters of the rotary evaporator are set as follows: the water bath temperature is 40-50° C., the vacuum degree is 100-300 mbar, and the rotation speed is 100-120 rpm. The combined extracts of step (1) are concentrated into a thick liquid by the rotary evaporator. The thick liquid is then pre-frozen at -40° C. for 3-4 h. The sample is then transferred to a freeze dryer, the cold trap temperature is set at -60° C., the tray temperature is set at 30° C., and freeze-dried for 24 h to obtain a gallnut extract, which is then stored at 4° C. for later use.

[0040] (3) The Chinese gallnut extract of step (2) is uniformly mixed with the above-mentioned auxiliary materials in a mass ratio of 1:(1-2), and a 30% ethanol solution is sprayed and continuously stirred until the material reaches a state of "forming a ball when held and falling apart when lightly touched", followed by manual granulation using a 14-mesh sieve, and the granules are dried at 60° C., and then sized using a 10-mesh sieve to obtain dark brown granules, thereby obtaining a traditional Chinese medicine preparation.

[0041] In the above step (3), the mass volume ratio of the total mass of the gallnut extract and the auxiliary materials to the ethanol solution is 100g:(2-5)mL; the optimal auxiliary materials are β-cyclodextrin and β-mannitol, and the mass ratio of β-cyclodextrin to β-mannitol is 1:(1-4).

[0042] The invention also discloses the application of the traditional Chinese medicine preparation in preparing a medicine product with liver protection effect.

[0043] Among them, the above-mentioned pharmaceutical product with liver protection effect can be a pharmaceutical product for alleviating or treating acute liver injury or liver fibrosis.

[0044] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments (wherein: Figure 1 The preparation of the Chinese medicine preparation provided by the present invention and its efficacy detection flow chart):

[0045] Example 1

[0046] The preparation method of the Chinese medicine preparation with liver protection effect in this embodiment comprises the following specific steps:

[0047] (1) After sieving the Gallic Acid, crush it into particles with a crusher. According to the mass volume ratio of Gallic Acid to n-hexane of 1 g:6 mL, weigh an appropriate amount of Gallic Acid particles and n-hexane, and place them in a round-bottom flask. After ultrasonic treatment for 1 h, separate the residue and the extract. Repeat the above extraction process for a total of 3 times, and then combine the extracts together.

[0048] (2) The parameters of the rotary evaporator are set as follows: the water bath temperature is 45° C., the vacuum degree is 150 mbar, and the rotation speed is 110 rpm. The combined extracts of step (1) are concentrated into a thick liquid by the rotary evaporator. The thick liquid is then pre-frozen at -40° C. for 3.5 h. The sample is then transferred to a freeze dryer, the cold trap temperature is set to -60° C., the tray temperature is set to 30° C., and freeze-dried for 24 h to obtain the gallnut extract, which is then stored at 4° C. for later use.

[0049] (3) The gallnut extract of step (2) and the above-mentioned auxiliary materials (the mass ratio of β-cyclodextrin to β-mannitol is 1:2) are uniformly mixed in a mass ratio of 1:1.5, and at the same time, a 30% ethanol solution with a mass volume fraction is sprayed (the mass volume ratio of the total mass of the gallnut extract and the auxiliary materials to the ethanol solution is 100 g:2.5 mL), and stirring is continued until the material reaches a state of "forming a ball when held and falling apart when touched lightly", and then manually granulating the particles using a 14-mesh sieve, drying the particles at 60°C, and then granulating the particles using a 10-mesh sieve to obtain dark brown particles, thereby obtaining a traditional Chinese medicine preparation.

[0050] Example 2

[0051] This example is the effect of the Chinese medicine preparation prepared in the above Example 1 on acute liver injury caused by acetaminophen.

[0052] In this embodiment, mice were used as experimental animals and the experiment was carried out under standard sanitary conditions. 30 mice were selected with a body weight of 16g ​​± 2g and randomly divided into 3 groups of 10 mice each, namely the control group (CW), the model group (MW), and the test group (OW). The mice in the test group (OW) were gavaged with Chinese medicine preparations every day at a dose of 100 mg / kg·bw; the mice in the control group (CW) and the model group (MW) received an equal volume of normal saline gavage; on the 14th day after the start of the experiment, the mice in the above-mentioned model group (MW) and the test group (OW) were given 300 mg / kg·bw acetaminophen solution by intraperitoneal injection to establish an acute liver injury model. 12 hours after modeling, all mice were killed and samples were collected for subsequent analysis, as follows.

[0053] (1) Effects of the Chinese medicine preparation of the present invention on serum interleukins, transaminases and antioxidant enzymes in mice with acute liver injury.

[0054] 1 mL of blood was collected from each mouse through eye sampling, and after standing at 4°C for 2 h, the supernatant was aspirated after centrifugation at 3000 rpm for 10 min. According to the instructions of the ELISA kit, the contents of interleukin-6, interleukin-10, interleukin-1β, tumor necrosis factor α (TNF-α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), total antioxidant capacity (T-AOC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were detected.

[0055] like Figure 2As shown, the serum ELISA results show that: the model group (MW) causes the antioxidant enzymes (SOD, GSH-Px) and total antioxidant capacity (T-AOC) in the mouse serum to be significantly reduced, the malondialdehyde (MDA) level to be increased, the transaminase (ALT, AST) level to be increased, and the inflammatory factor (IL-6, IL-10, IL-1β, TNF-α) level to be increased, indicating that acetaminophen causes certain damage to the mouse liver, resulting in a decrease in the body's antioxidant capacity and an obvious inflammatory response; in the test group (OW), the antioxidant enzymes (SOD, GSH-Px) and total antioxidant capacity (T-AOC) in the mouse serum are significantly increased, the malondialdehyde (MDA) level to be decreased, the transaminase (ALT, AST) level to be decreased, and the inflammatory factor (IL-6, IL-10, IL-1β, TNF-α) level to be decreased, indicating that the Chinese medicine preparation provided by the present invention can effectively combat the acute liver injury caused by acetaminophen, and play a certain protective role by enhancing the antioxidant capacity and reducing the inflammatory response.

[0056] (2) Effects of the Chinese medicine preparation of the present invention on the intestinal flora structure of mice with acute liver injury.

[0057] 0.2 g of intestinal contents were taken from each group of mice (control group, model group and experimental group) as samples, and total DNA was extracted. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using specific primers, and the amplified PCR products were treated with a kit to construct a sequencing library. After the library construction was completed, these libraries were used for sequencing to generate a large amount of sequence data, and then bioinformatics analysis was performed on these sequence data to evaluate and compare the relative abundance and changes of dominant strains in the intestines of each group of mice under different treatment conditions at different taxonomic levels.

[0058] like Figure 3As shown, (i) at the phylum level: at the phylum level, compared with the CW group, the relative abundance of Actinobacteriota (p<0.05) and Firmicutes_B_370539 increased in the MW group, but decreased in the OW group; the abundance of Desulfobacteriota-I decreased in the MW group, but increased in the OW group. (ii) At the genus level: compared with the CW group, the abundance of Pelethenecus, Odoribacter and Mailhella decreased in the MW group, but increased in the OW group. The abundance of Faecalibaculum, UBA3789 (p<0.05) and Jeotgalicoccus_A_310962 (p<0.05) in the MW group was higher than that in the CW group, and the abundance in the OW group was higher than that in the MW group. The abundance of several genera, including s_E(p<0.05), Cryptobacteroides, Muribaculum, Corynebacterium, Lactococcus, Rikenella, Alloprevotella, Bacteroides_H, Prevotella, Phocaeicola, Alistipes and Turicibacter, in the MW group was higher than that in the CW group, and lower than that in the OW group. Existing studies have shown that Odoribacter is a beneficial bacterium, and its increased abundance is related to anti-inflammatory effects; Rikenella, Phocaeicola, Alloprevotella and other genera are harmful bacteria associated with a variety of diseases; this indicates that acetaminophen treatment leads to intestinal flora disorder in mice, and the intestinal flora structure can be restored after treatment with the Chinese medicine preparation provided by the present invention, which can reduce inflammation by promoting the growth of beneficial bacteria and inhibiting the growth of harmful bacteria.

[0059] Example 3

[0060] This example is a test on the protection of the Chinese medicine preparation prepared in the above example 1 against chronic liver damage.

[0061] This embodiment uses mice as experimental animals, and the test is carried out under standard sanitary conditions. 30 mice are selected, with a body weight of 16g ​​± 2g, and are randomly divided into 3 groups of 10 mice in each group, namely the control group (CTRL), the model group (CCl4), and the test group (CCl4 + RM). The mice in the test group (CCl4 + RM) are gavaged with Chinese medicine preparations every day, with a dose of 100mg / kg·bw; the mice in the control group (CTRL) and the model group (CCl4) receive an equal volume of normal saline gavage. From the beginning of the test, the model group (CCl4) and the test group (CCl4 + RM) mice are intraperitoneally injected with 2mL / kg·bw of 10% carbon tetrachloride olive oil solution every 3 days, for 4 weeks, 10 injections in total, and a total of 28 days; at the same time, the mice in the blank control group (CTRL) receive the same dose of olive oil intraperitoneal injection; 24h after the last administration, all mice are killed, and blood and liver samples are collected for subsequent analysis.

[0062] (1) Effects of the Chinese medicine preparation of the present invention on the liver tissue morphology of mice with liver fibrosis.

[0063] Take the same position of about 1cm in the right lobe of the liver of each group of mice 3 The tissues of different sizes were fixed with 4% paraformaldehyde solution, then routinely dehydrated and embedded in paraffin. The embedded tissues were cut into slices with a thickness of about 3 μm; these slices were dewaxed with xylene, then dehydrated step by step with ethanol, and then HE staining, Sirius red staining and Masson staining were performed on the slices to show the pathological changes of liver tissue from different angles. After staining, they were dehydrated step by step with ethanol again, transparentized with xylene, and finally sealed with neutral gum. The prepared slices were observed under an electron microscope and the pathological changes of liver tissue were recorded.

[0064] like Figure 4 As shown, the results show that: HE staining shows that after the model group (CCl4) is treated, the liver sinusoidal tissue structure is significantly damaged, accompanied by obvious inflammatory cell aggregation and hepatocyte necrosis; Sirius red staining and Masson staining further show that after the CCl4 group is treated, there is significant collagen fiber precipitation in the liver tissue, which indicates that CCl4 has caused serious damage to the liver tissue structure and there is obvious fibrosis in the liver. Compared with the model group (CCl4), the test group (CCl4+RM) samples treated with the Chinese medicine preparation of the present invention showed a significantly improved effect, with reduced inflammatory cell aggregation, alleviated tissue structure damage, and significantly reduced amount of deposited collagen fibers; this shows that the Chinese medicine preparation of the present invention can effectively reduce liver damage and its fibrosis process caused by CCl4, has the ability to repair damaged liver cell structure and inhibit excessive deposition of collagen fibers, and shows the potential for protecting and repairing the liver.

[0065] (2) Effects of the Chinese medicine preparation of the present invention on serum interleukins, transaminases and antioxidant enzymes in mice with liver fibrosis.

[0066] 1 mL of blood was collected from each mouse by eye sampling, and after standing at 4°C for 2 h, the supernatant was aspirated after centrifugation at 3000 rpm for 10 min. According to the instructions of the ELISA kit, the levels of interleukin-6, interleukin-10, interleukin-1β, tumor necrosis factor α (TNF-α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), total antioxidant capacity (T-AOC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were detected.

[0067] like Figure 5 As shown, the results show that the model group (CCl4) significantly reduced the antioxidant levels (SOD, GSH-Px, T-AOC) and transaminase levels (ALT, AST) in the serum of mice, while the levels of inflammatory factors (interleukin 6, interleukin 10, interleukin 1β, tumor necrosis factor α) increased; the antioxidant levels (SOD, GSH-Px, T-AOC), transaminase levels (ALT, AST) and inflammatory factor levels (interleukin 6, interleukin 10, interleukin 1β, tumor necrosis factor α) in the serum of mice in the test group (CCl4+RM) were all restored. This shows that the Chinese medicine preparation of the present invention can enhance the antioxidant capacity in mice, effectively regulate the inflammatory response, reduce liver damage caused by carbon tetrachloride, thereby improving liver function, and showing its potential mechanism for liver protection.

[0068] (3) Effect of the Chinese medicine preparation of the present invention on the expression of Nrf2 signaling pathway in mice with liver fibrosis.

[0069] The liver tissue samples of the mice in the above groups were taken, ground with Trizol extraction solution, and operated on ice to maintain the integrity of RNA. High-quality RNA was extracted through chloroform separation, isopropanol precipitation, and ethanol washing steps. The extracted RNA was reverse transcribed into cDNA by RT-PCR, and then quantitatively analyzed by qPCR to evaluate the expression level of the target gene.

[0070] like Figure 6As shown, the results show that: The results show that: The model group (CCl4) significantly reduced the expression levels of Nrf2 / HO-1 signaling pathway related genes (including Nrf2, HO-1, NQO-1) in the mouse liver, and increased the expression levels of apoptosis regulatory genes BAX and BCL2, which indicates that after treatment with the model group (CCl4), not only the liver's antioxidant defense system was weakened, but also the apoptosis process of hepatocytes was promoted. In the test group (CCl4+RM), the expression levels of Nrf2, HO-1, NQO-1, BAX and BCL2 genes in the mouse liver were significantly restored. This shows that the Chinese medicine preparation of the present invention not only improves the antioxidant capacity in the mouse liver, but also reduces the apoptosis of hepatocytes, thereby effectively alleviating the liver damage caused by carbon tetrachloride, that is, the Chinese medicine preparation of the present invention can effectively activate the Nrf2 signaling pathway, enhance the antioxidant capacity of the liver, and reduce the apoptosis of hepatocytes.

[0071] Take the liver tissue samples of the above-mentioned groups of mice, add RIPA lysis buffer for grinding, and operate on ice to extract total protein. After determining and adjusting the protein concentration using the BCA method, add loading buffer and heat in a boiling water bath for 10 minutes to denature the protein, then separate the protein by 10% SDS-PAGE electrophoresis, and transfer the protein using a PVDF membrane. The membrane was placed in 5% skim milk, blocked for 1 hour at room temperature, incubated with the corresponding primary antibody at 4°C overnight, washed twice, and then incubated with the secondary antibody at room temperature for 1 hour. Finally, the protein bands were displayed using ECL development technology, and the grayscale values ​​of the protein bands were measured using Image J software.

[0072] like Figure 7 As shown in the figure, it can be seen that the model group (CCl4) significantly reduced the expression level of Nrf2 / HO-1 signaling pathway related proteins (including Nrf2, HO-1 and NQO-1) in the mouse liver, and the decrease in the expression of this signaling pathway was closely related to the weakening of the mouse's antioxidant capacity; the experimental group (CCl4+RM) significantly restored the expression level of Nrf2, HO-1 and NQO-1 proteins in the mouse liver; this indicates that the traditional Chinese medicine preparation prepared by the present invention can restore the mouse's antioxidant capacity to a certain extent and reduce the apoptosis of liver cells.

[0073] (4) Effects of the Chinese medicine preparation of the present invention on liver metabolomics in mice with liver fibrosis.

[0074] Metabolites with significant differences between the groups were considered as potential biomarkers.We screened metabolites with significant differences between the control group (CTRL) and the model group (CCl4) and between the model group (CCl4) and the experimental group (CCl4+RM).

[0075] like Figure 8As shown in the results, a total of 10 metabolites with significant differences were found, including morphine N-oxide, leucine-glutamate (Leu-Glu), bisindolylmaleimide VIII (bis VIII), 5-demethylnobiletin, irigenin, 2-hydroxy-2-methylbutyric acid, 3-isoxazolecarboxamide, N,N-dicyclohexyl-5-cyclopropyl, butaprost, and N-[3-(2-oxopyrrolidin-1-yl)propyl]acetamide. Among them, bisindolylmaleimide VIII, irisin and butaprost are considered to have important physiological significance. Bisindolylmaleimide VIII can inhibit the degradation of Nrf2, thereby stabilizing and enhancing the function of the Nrf2 signaling pathway; irisin can directly promote the expression of the Nrf2 signaling pathway, further enhancing the body's antioxidant capacity; these two metabolites act together on the Nrf2 signaling pathway through different mechanisms, helping to enhance the liver's antioxidant defense system; butaprost can inhibit the deposition of collagen and reduce tissue fibrosis.

[0076] Depend on Figure 8 It can be seen that: compared with the model group (CCl4), after treatment with the test group (CCl4+RM), the content of irisin and butaprost increased, while the content of bisindolylmaleimide VIII decreased, and it was observed that collagen deposition decreased; Nrf2 signaling pathway expression was upregulated, and the body's antioxidant capacity increased. This shows that the Chinese medicine preparation of the present invention directly promotes the activation of the Nrf2 signaling pathway and inhibits collagen deposition by improving the levels of irisin and butaprost; at the same time, by reducing the content of bisindolylmaleimide VIII, it indirectly supports the stability and activity of the Nrf2 signaling pathway, thereby reducing liver fibrosis in mice caused by carbon tetrachloride. It is thus shown that the Chinese medicine preparation provided by the present invention not only helps to restore the normal function of the liver, but also effectively alleviates oxidative stress and fibrosis, thereby improving liver health.

[0077] (5) Effects of the Chinese medicine preparation of the present invention on the intestinal flora of mice with liver fibrosis.

[0078] 0.2 g of intestinal contents were taken from each group of mice (control group, model group and experimental group) as samples, and total DNA was extracted. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using specific primers, and the amplified PCR products were treated with a kit to construct a sequencing library. After the library construction was completed, these libraries were used for sequencing to generate a large amount of sequence data, and then bioinformatics analysis was performed on these sequence data to evaluate and compare the relative abundance and changes of dominant strains in the intestines of each group of mice under different treatment conditions at different taxonomic levels.

[0079] like Fig. 9 As shown in the figure, compared with the model group (CCl4), the relative abundance of Akkermansia, Dubosiella and UBA_3263 at the genus level in the experimental group (CCl4+RM) was significantly increased. To further explore the relationship between these genera and key metabolites, Spearman correlation analysis was performed.

[0080] The results showed that Akkermansia was significantly negatively correlated with bisindolylmaleimide VIII, but significantly positively correlated with butaprost and irisin; Dunovettia was significantly positively correlated with butaprost; UBA_3263 was also significantly positively correlated with butaprost, but significantly negatively correlated with bisindolylmaleimide VIII. This shows that the Chinese medicine preparation provided by the present invention can significantly restore the relative abundance of beneficial bacteria such as Akkermansia, Dunovettia and UBA_3263, affect the content of key metabolites in the liver by regulating the intestinal-liver axis, further enhance the body's antioxidant capacity and anti-inflammatory response, reduce the level of inflammatory factors, and promote liver cell repair and regeneration.

[0081] The above is a detailed introduction to a Chinese medicine preparation with liver-protecting effect disclosed in the present invention, as well as its preparation method and application. Specific examples are used herein to illustrate the principles and implementation methods of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A Chinese medicine preparation having a liver-protecting effect, characterized in that: The traditional Chinese medicine preparation is prepared from a gallnut extract, an auxiliary material and an ethanol solution; the mass ratio of the gallnut extract to the auxiliary material is 1: (1-2), the mass volume ratio of the total mass of the gallnut extract and the auxiliary materials to the ethanol solution is 100g: (2~5)mL.

2. A Chinese medicine preparation with liver protection effect according to claim 1, characterized in that: The auxiliary materials are β-cyclodextrin and β-mannitol, and the mass ratio of β-cyclodextrin to β-mannitol is 1: (1～4)。 3. A Chinese medicine preparation with liver protection effect according to claim 2, characterized in that: The mass ratio of the gallnut extract to the auxiliary material is 1:1.5; the mass volume ratio of the total mass of the gallnut extract and the auxiliary material to the ethanol solution is 100g:2.5mL; the mass ratio of β-cyclodextrin to β-mannitol is 1:

2.

4. The method for preparing the Chinese medicinal preparation with liver protection effect according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) After sieving the Chinese gallnut to remove impurities, crush it into particles with a crusher, take an appropriate amount of the Chinese gallnut particles and place them in a round-bottom flask, then add n-hexane to the round-bottom flask, and after ultrasonic treatment for 1 hour, separate the residue and the extract, repeat the above extraction process for a total of 3 times, and then combine the extracts together; (2) using a rotary evaporator to concentrate the combined extracts from step (1) into a thick liquid, and then freeze-drying the thick liquid to obtain a gallnut extract, and storing it at 4° C. for later use; (3) The Chinese gallnut extract of step (2) is uniformly mixed with the above-mentioned auxiliary materials, and ethanol solution is sprayed and continuously stirred until the material reaches a state of "forming a mass when held and loosening when touched lightly", and then manually granulated using a 14-mesh sieve, and the granules are dried at 60° C., and then sized using a 10-mesh sieve to obtain dark brown granules, thereby obtaining a traditional Chinese medicine preparation.

5. The method for preparing the Chinese medicinal preparation having liver protection effect according to claim 4, characterized in that: In step (1), the mass volume ratio of the gallnut particles to n-hexane is 1 g: (3-10) mL.

6. The method for preparing the Chinese medicinal preparation having liver-protecting effect according to claim 4, characterized in that: In step (2), the process parameters of the rotary evaporation are: water bath temperature of 50-60°C, vacuum degree of 100-300 mbar, and rotation speed of 100-120 rpm; the freezing process is: pre-freezing at -40°C for 3-4 hours, then transferring the sample to a freeze dryer, setting the cold trap temperature to -60°C, the tray temperature to 30°C, and freeze drying for 24 hours.

7. The method for preparing the Chinese medicinal preparation having liver protection effect according to claim 4, characterized in that: In step (3), the mass volume fraction of the ethanol solution is 30%.

8. Use of the traditional Chinese medicine preparation according to any one of claims 1 to 3 in the preparation of a pharmaceutical product having a liver-protecting effect.

9. The use according to claim 8, characterized in that: The pharmaceutical product with liver protection effect is a pharmaceutical product for alleviating or treating acute liver injury or liver fibrosis.

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