Anti-hepatic fibrosis plant extract as well as preparation method and application thereof

Abstract

The invention discloses an anti-hepatic fibrosis plant extract, and relates to the technical field of plant extraction medicines, and the plant extract is obtained by mixing and extracting corydalis bungeana, herpetospermum pedunculosum, hypecoum erectum, bergenia purpurascens and myrobalan. According to the anti-hepatic fibrosis Tibetan medicine formula, the complex physiological microenvironment of hepatic lesions is accurately reproduced through an animal model, and the metabolic process, the distribution condition and the effect on a target organ of a medicine in a living body are researched, so that the anti-hepatic fibrosis Tibetan medicine formula is provided; more scientific and powerful theoretical support and experimental basis are expected to be provided for further development and utilization of Tibetan medicine compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of plant-extracted drugs, and particularly relates to an anti-hepatic fibrosis plant extract, a preparation method thereof, and an application thereof. Background Art

[0002] Hepatic fibrosis is a pathological change caused by chronic liver damage due to various reasons, manifested as excessive abnormal deposition of extracellular matrix components in the liver, which affects the function of the liver and is an inevitable stage for chronic liver diseases to develop into liver cirrhosis. It is currently believed that hepatic fibrosis may still be reversible to normal, while liver cirrhosis is not. At present, the focus of research is on the interactions between molecules, between molecules and cells, and between cells. Although there have been some progress in diagnosis and treatment, there is still a lack of definite and effective drugs.

[0003] Currently, the main methods for treating hepatic fibrosis are: treating the primary disease and removing the causative factors. There are numerous literature reports that after removing the causative factors, hepatic fibrosis caused by various liver diseases can be reversed, but it cannot completely treat liver cirrhosis and hepatic fibrosis, and the effect is relatively slow. Weakening inflammation and immune responses. During the treatment of HCV with IFN-α, it has been observed that even if patients do not respond to virus suppression, there can be a moderate anti-fibrosis effect, suggesting that the anti-inflammatory effect of IFN-α may have a certain anti-fibrosis effect; however, the effect is not obvious.

[0004] So far, Western medicine has not had a very good effect on treating hepatic fibrosis and liver cirrhosis, and chemotherapy causes great harm to the human body, mainly manifested in the damage to the human immune system. In recent years, in the scientific research and clinical practice of traditional Chinese medicine, it has been found that traditional Chinese medicine has certain advantages in the treatment of various liver diseases. There are many patents disclosing the use of drugs for promoting blood circulation and removing stasis to treat hepatic fibrosis and liver cirrhosis, but the effect is still not particularly ideal.

[0005] The development and utilization of Tibetan medicine compounds is one of the hot topics in the current field of medical research, and pharmacodynamic research plays a crucial role in it. This application accurately reproduces the complex physiological microenvironment of liver lesions with an animal model, and provides a Tibetan medicine formula for anti-hepatic fibrosis by studying the metabolic process, distribution, and effects on target organs of the drug in vivo, in order to provide more scientific and powerful theoretical support and experimental basis for the further development and utilization of Tibetan medicine compounds. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an anti-hepatic fibrosis plant extract, a preparation method thereof, and an application thereof.

[0007] The purpose of the present invention is achieved by the following technical solutions: An anti-hepatic fibrosis plant extract, which is obtained by mixing and extracting Corydalis bungeana Turcz., Herba Herpetospermi, Hypecoum erectum L., Bergenia purpurascens (Hook. f. et Thoms.) Engl., and Terminalia chebula Retz.

[0008] The extract inhibits the expression of COl1A1, TGF-β11, and α-SMA proteins.

[0009] By weight, it includes 10-40 parts of Corydalis bungeana Turcz., 10-40 parts of Herba Herpetospermi, 10-40 parts of Terminalia chebula Retz., 10-30 parts of Hypecoum erectum L., and 10-20 parts of Bergenia purpurascens (Hook. f. et Thoms.) Engl.

[0010] Preferably, the mass ratio of Corydalis bungeana Turcz. : Herba Herpetospermi : Hypecoum erectum L. : Bergenia purpurascens (Hook. f. et Thoms.) Engl. : Terminalia chebula Retz. is 2:1:2:1.5:2.

[0011] The second aspect of the present invention provides a preparation method of an anti-hepatic fibrosis plant extract, including the following steps: S1. Take Corydalis bungeana Turcz., Herba Herpetospermi, Hypecoum erectum L., Bergenia purpurascens (Hook. f. et Thoms.) Engl., and Terminalia chebula Retz., and powder them to obtain a powder material; S2. Carry out boiling water reflux extraction on the powder material to obtain an extract paste; S3. Purify the extract paste to obtain an extract.

[0012] Specifically, the boiling water reflux extraction in S2 is as follows: soak the powder material in normal temperature water for 1 h, then heat it to boiling and carry out reflux extraction for 1 h, and extract 2 times; the extract is filtered and concentrated to obtain an extract paste.

[0013] The ratio of the powder material to normal temperature water is 1:10.

[0014] Specifically, the purification in S3 is as follows: freeze-dry the extract paste to obtain a powdered extraction product, add water to make a sample loading solution, and use the D101 macroporous resin method to enrich the active ingredients of the sample loading solution.

[0015] The specific steps of the D101 macroporous resin method are as follows: slowly pour the sample loading solution into the column, with an elution flow rate of 3 mL / min, let it stand and adsorb for 2 h after loading the sample; use ultrapure water for elution until the eluate is colorless; prepare a 10%-50% ethanol aqueous solution, slowly add the ethanol aqueous solution, elute until the eluate is colorless, collect the eluate and concentrate it to a non-alcohol extract paste state, and then freeze-dry to obtain an extract.

[0016] The third aspect of the present invention provides the application of an anti-hepatic fibrosis plant extract in liver protection and liver disease treatment.

[0017] The beneficial effects of the present invention are: The extracts of Corydalis bungeana Turcz., Herba Herpetospermi, Hypecoum erectum L., Bergenia purpurascens (Hook. f. et Thoms.) Engl., and Terminalia chebula Retz. provided in this application can affect the expression of fibrosis marker proteins such as COl1A1, α-SMA, and TGF-β1, thereby achieving a therapeutic effect.

[0018] This application precisely reproduces the complex physiological microenvironment of liver lesions using an animal model, studies the metabolic process, distribution, and effects on target organs of drugs in vivo, and provides a Tibetan medicine formula for anti-hepatic fibrosis, aiming to provide more scientific and powerful theoretical support and experimental basis for the further development and utilization of Tibetan medicine compounds. Brief Description of the Drawings

[0019] Figure 1 It is a scatter plot of the cytotoxicity results of each component on three types of cells detected by the CCK-8 method; Figure 2 It is a graph of the periodic change in body weight; Figure 3 It is a graph of liver coefficients; Figure 4 It is a graph of the coefficients of the heart, spleen, lung, and kidney; Figure 5 It is a graph of liver tissue; Figure 6 It is a HF staining graph of rat liver tissue; Figure 7 It is a Masson staining graph of rat liver tissue; Figure 8 It is a blood biochemical analysis graph of ALT, AST, ALB, and T-Bil in rats; Figure 9 It is the biochemical analysis results of HA and LN in rats; Figure 10 It is the biochemical analysis results of PCⅢ and CⅣ in rats; Figure 11 It is a HYP analysis graph of rats; Figure 12 It is the WB gray scale analysis of COl1A1, α-SMA, and TGF-β1; Figure 13 It is the WB result graph of COl1A1, α-SMA, and TGF-β1; Detailed Embodiments

[0020] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present invention.

[0021] Embodiment 1 Take 20 g of Corydalis edulis, 10 g of Herpetospermum pedunculosum, 20 g of Hypecoum erectum, 15 g of Bergenia purpurascens, and 20 g of Terminalia chebula. Use boiling water reflux extraction for the above plant combination, namely the Five-Ingredient Corydalis Powder (WWZJS). First, powder the medicinal materials, pour them into normal temperature water according to the material-liquid ratio of 1:10, soak for 1 h, then heat to boiling and reflux extract for 1 h, extract twice, filter the insoluble substances, and collect the filtrate and concentrate it to obtain an extract paste.

[0022] Freeze-dry the extract paste to obtain a powdered extraction product, add water to make a sample solution, and use the D101 macroporous resin method to enrich the active ingredients respectively. Slowly pour the sample solution into the column, maintain the elution flow rate at 3 mL / min, let it stand and adsorb for 2 h after loading the sample, use ultrapure water for elution until the effluent is colorless, prepare a 10% ethanol aqueous solution, slowly add the 10% ethanol aqueous solution, elute until the effluent is colorless, collect the effluent and concentrate it to a non-alcoholic extract paste state, and weigh the obtained extract after freeze-drying.

[0023] Example 2 Take 20 g of Corydalis edulis, 10 g of Herpetospermum pedunculosum, 20 g of Hypecoum erectum, 15 g of Bergenia purpurascens, and 20 g of Terminalia chebula. Use boiling water reflux extraction for the above plant combination, namely the Five-Ingredient Corydalis Powder (WWZJS). First, powder the medicinal materials, pour them into normal temperature water according to the material-liquid ratio of 1:10, soak for 1 h, then heat to boiling and reflux extract for 1 h, extract twice, filter the insoluble substances, and collect the filtrate and concentrate it to obtain an extract paste.

[0024] Freeze-dry the extract paste to obtain a powdered extraction product, add water to make a sample solution, and use the D101 macroporous resin method to enrich the active ingredients respectively. Slowly pour the sample solution into the column, maintain the elution flow rate at 3 mL / min, let it stand and adsorb for 2 h after loading the sample, use ultrapure water for elution until the effluent is colorless, prepare a 30% ethanol aqueous solution, slowly add the 30% ethanol aqueous solution, elute until the effluent is colorless, collect the effluent and concentrate it to a non-alcoholic extract paste state, and weigh the obtained extract after freeze-drying.

[0025] Example 3 Take 20 g of Corydalis edulis, 10 g of Herpetospermum pedunculosum, 20 g of Hypecoum erectum, 15 g of Bergenia purpurascens, and 20 g of Terminalia chebula. Use boiling water reflux extraction for the above plant combination, namely the Five-Ingredient Corydalis Powder (WWZJS). First, powder the medicinal materials, pour them into normal temperature water according to the material-liquid ratio of 1:10, soak for 1 h, then heat to boiling and reflux extract for 1 h, extract twice, filter the insoluble substances, and collect the filtrate and concentrate it to obtain an extract paste.

[0026] Freeze-dry the extract paste to obtain a powdered extraction product, add water to make a sample solution, and use the D101 macroporous resin method to enrich the active ingredients respectively. Slowly pour the sample solution into the column, maintain the elution flow rate at 3 mL / min, let it stand and adsorb for 2 h after loading the sample, use ultrapure water for elution until the effluent is colorless, prepare a 50% ethanol aqueous solution, slowly add the 50% ethanol aqueous solution, elute until the effluent is colorless, collect the effluent and concentrate it to a non-alcoholic extract paste state, and weigh the obtained extract after freeze-drying.

[0027] Example 4 The CCK-8 method was used to detect the toxicity of the extracts obtained in Examples 1-3 to HSC-T6 cells, LX-2 cells, and THLE-3 cells. In the experiment, the sterile normal saline was less than 6%, and the DMSO concentration was less than 0.08%. The control group only adopted boiling water reflux extraction of the plant composition without purification by the D101 macroporous resin method (total extract).

[0028] Among them, Table 1 shows the IC50 values of the extracts on HSC-T6 and LX-2 cells. It can be seen that the inhibitory effect of the purified extract on the proliferation of hepatic stellate cells is significantly enhanced, and the drug concentrations required to inhibit the two cells by half for each component are reduced by multiples (P < 0.001). The scatter plot of the results of the CCK-8 method for detecting the toxicity of each component to the three cells is as Figure 1 shown. Overall, when the purified drug concentration exceeds 60 mg / mL, the survival rates of HSC-T6 and LX-2 cells are both lower than 50% (i.e., IC50), indicating that each purified part has an inhibitory effect on the proliferation of hepatic stellate cells. However, the inhibitory trend of the 10% purified product weakens after exceeding 60 mg / mL and does not even increase with the increase in concentration ( Figure 1 A). In the effect on THLE-3 cells, the 10% and 30% purified products have relatively less impact on the cells compared with the 50% purified product.

[0029] Table 1 IC50 values of each purified extract on HSC-T6 and LX-2 cells Note: A differential analysis was performed for each column with the non-purified extract.

[0030] Example 5 Using an animal model to study the effect of plant extracts on liver fibrosis 1. Behavioral and physiological observations and evaluations of SD rats After 80 male SD rats were housed in the animal house for one week, they were randomly divided into six groups: blank group (Control), model group (Model), Silymarin group, low-dose group (LD), medium-dose group (MD), and high-dose group (HD) of WWZJS, with 12 rats in each group. Modeling was performed at a dose of 0.3 mL / kg (0.2 mL / kg for the first time and 0.3 mL / kg for the second time). Starting from the fifth week, the drugs were administered by gavage. The volume of gavage for each rat was within 1 ml / 100g each time, with a maximum of no more than 4 ml / 100g, and the modeling was continued simultaneously. The rats were weighed weekly, and changes in their behavior, hair color, appearance, etc. were observed. The results are shown in Table 2.

[0031] Table 2 Administration Schedule for Modeling Rat Liver Fibrosis After administration in the 8th week, after fasting for 12 h, samples were taken and weighed. The heart, liver, spleen, lung, and kidney were each taken and weighed and recorded.

[0032] The results showed that in the early stage of modeling, all SD rats had white and shiny hair, clean claws, ears, etc.; good mental state, active movement; normal diet, normal excretion volume and color. There was no obvious abnormality after the first modeling, but after the second modeling, there was an obvious stop in weight gain, and even a decrease in the weight of some individuals; after continuous modeling, the weight recovered, but the body size and weight were generally significantly smaller than those in the Contral group, and the hair color was yellowish, with a hunched-back phenomenon, and there was a situation where the feces of some individuals were unformed. Administration started in the fifth week, and the weight cycle change chart showed ( Figure 2 ), after administration of the positive drug Silymarin, the weight increased significantly, and the weight increased steadily after overall administration. The overall weight increase in the Model group was the smallest. The hunched-back state in the administration group improved significantly compared with the Model group, and the hair color became relatively white.

[0033] When taking samples, the weight of each rat was measured, and the heart, liver, spleen, lung, and kidney tissues were taken and weighed to calculate the liver coefficient. The liver coefficient in the Model group was significantly different from that in other groups (P < 0.001) ( Figure 3 ), but for the coefficients of other organs, there was no significant difference among the six groups (P>0.05) ( Figure 4 ). In the differential analysis in the figure, # indicates comparison between the Mode group and the Contral group; * indicates comparison between the administration group and the Model group. The numbers 1 - 3 marked by the symbols indicate P < 0.05, P < 0.01, and P < 0.001 in sequence, and ns indicates P>0.05.

[0034] Observing the morphological structure of the liver tissue, it could be seen that the liver surface in the Model group was granular and not smooth, and the liver lobes were thicker compared with the blank group, with obvious differences. In the administration group, the surface was relatively smooth compared with the model group, and the pathological features were significantly alleviated ( Figure 5 ).

[0035] The pathological sections of the liver tissues of each group were stained with HE and Masson. The results of HE staining showed ( Figure 6 ), in the Model group, the hepatic lobule structure was severely damaged, and the hepatocytes showed moderate and severe hydropic degeneration, inflammatory cell infiltration, hepatocyte degeneration, and necrosis. In the administration group, the hepatocytes were arranged neatly in cords and the structure was complete, indicating obvious improvement after administration. The results of Masson staining ( Figure 7The blue color indicates collagen fiber tissue hyperplasia. In the Model group, a large number of fibrous septa and false lobules were formed, indicating successful modeling. The blue collagen fibers in the four drug administration groups were significantly reduced, and the fibrous collagen tissue hyperplasia in the portal areas of some hepatic lobules was significantly reduced. The structure was basically no different from that of the blank group and was relatively intact. Combining the results of the two staining methods, it can be proved that the pathological changes of the liver tissue were improved after the intervention of Silymarin and WWZJS.

[0036] Example 6 Effect of plant extracts on liver injury and liver fibrosis in rats.

[0037] Biochemical analysis of ALT, AST, ALB, and T-Bil related to liver injury was performed on the blood of HF (hepatic fibrosis) rats ( Figure 8 ). There were significant differences in the liver injury indicators of the drug administration groups compared with the Model group. In the differential analysis in the figure, # indicates the comparison between the Mode group and the Contral group; * indicates the comparison between the drug administration group and the Model group. The symbol markings 1-3 in sequence indicate P < 0.05, P < 0.01, and P < 0.001, and ns indicates P > 0.05.

[0038] Analysis and detection results of the four liver fibrosis indicators (HA, PCⅢ, CⅣ, and LN) and the HYP analysis results showed that ( Figures 9 - 11 ) there were significant differences between the drug administration groups and the Model group, and the corresponding differences increased after increasing the dose, indicating that the modeling in the model group was successful and WWZJS had preventive and therapeutic effects on CCl 4 -induced liver fibrosis in SD rats. In the differential analysis in the figure, # indicates the comparison between the Mode group and the Contral group; * indicates the comparison between the drug administration group and the Model group. The symbol markings 1-3 in sequence indicate P < 0.05, P < 0.01, and P < 0.001, and ns indicates P > 0.05.

[0039] The WB experiment was performed to detect the expression of proteins related to liver fibrosis. The experimental results showed that the expressions of these fibrosis marker proteins such as COl1A1, α-SMA, and TGF-β1 were significantly reduced after the intervention of WWZJS ( Figures 12 - 13 ), proving the effectiveness of the drug.

[0040] From the above data, it can be seen that the anti-liver fibrosis plant extract provided by the present invention has a good effect on the treatment of the disease.

[0041] The above are only the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein, and should not be regarded as excluding other embodiments. Instead, it can be used in various other combinations, modifications, and environments, and can be changed within the scope of the concept described herein through the above teachings or the technology or knowledge in related fields. As long as the changes and variations made by those skilled in the art do not depart from the spirit and scope of the present invention, they should all be within the protection scope of the appended claims of the present invention.

Claims

1. An anti-liver fibrosis plant extract, characterized in that: The plant extract is obtained by mixed extraction of Corydalis tinctorius, Melon seeds, Fennel, Bergenia bergensis and Terminalia chebula.

2. The plant extract according to claim 1, characterized in that: The extract inhibits the expression of CO11A1 protein.

3. The plant extract according to claim 1, characterized in that: The extract inhibits α-SMA protein expression.

4. The plant extract according to claim 1, characterized in that: The extract inhibits TGF-β1 protein expression.

5. The plant extract according to claim 1, characterized in that The invention comprises, by weight, 10 to 40 parts of Corydalis chinensis, 10 to 40 parts of Terminalia chebula, 10 to 40 parts of Melon seeds, 10 to 30 parts of Fennel and 10 to 20 parts of Bergenia rapa.

6. The plant extract according to claim 1, characterized in that: The mass ratio of Corydalis tinctorius: Melon seeds: Fennel: Bergenia oleifera: Terminalia chebula is 2:1:2:1.5:

2.

7. A method for preparing an anti-liver fibrosis plant extract, characterized in that: The following steps are involved: S1. Grind corydalis, melon seeds, fennel, bergenia, and terminalia chebula into powder to obtain powder; S2, subjecting the powder to boiling water reflux extraction to obtain an extract; S3, purifying the extract to obtain the extract.

8. The preparation method according to claim 7, characterized in that: The boiling water reflux extraction in S2 is specifically as follows: soak the powder in room temperature water for 1 hour, then heat to boiling and reflux extract for 1 hour, extracting twice; the extract is filtered and concentrated to obtain an extract; the ratio of the powder to room temperature water is 1:

10.

9. The preparation method according to claim 7, characterized in that: The purification described in S3 is specifically as follows: freeze-drying the extract to obtain a powdered extract product, adding water to prepare a loading solution, and enriching the loading solution for active ingredients using a D101 macroporous resin method.

10. The preparation method according to claim 9, characterized in that: The specific steps of the D101 macroporous resin method are: slowly pouring the sample solution into the column, with an elution flow rate of 3 mL / min, and allowing to stand for adsorption for 2 hours after loading; eluting with ultrapure water until the effluent is colorless; preparing a 10%-50% ethanol aqueous solution, slowly adding the ethanol aqueous solution, eluting until the effluent is colorless, collecting the effluent and concentrating it to an alcohol-free extract, and then freeze-drying to obtain the extract.

11. Use of the anti-liver fibrosis plant extract according to any one of claims 1 to 6 in treating liver fibrosis and protecting the liver.

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