Application of Miao medicine botrychium ternatum and extract of Miao medicine botrychium ternatum in preparation of medicine for treating liver function impairment

Abstract

The invention relates to the technical field of medicines, in particular to application of Miao medicine botrychium ternatum and an extract thereof in preparation of liver protection medicines. The invention provides the following two applications of botrychium ternatum and an extract thereof: (1) the botrychium ternatum and the extract thereof are used for preventing / treating cell injury and tissue injury caused by oxidation and inflammation; and (2) preventing / treating acute and chronic liver injury. It is fully verified that the botrychium ternatum extract has an obvious liver protection effect and dose dependence and can obviously protect liver injury. According to the botrychium ternatum and the specific botrychium ternatum extract disclosed by the invention, the release of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) can be reduced, the necrosis degree of liver cells can be remarkably reduced, and the inflammation infiltration degree can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the application of Botrychium ternatum (Thunb.) Sw. and its extract in the preparation of drugs for liver function impairment. BACKGROUND

[0002] Liver is one of the main organs for regulating various physiological functions in the human body. According to traditional Chinese medicine, liver governs blood and regulates the flow of qi. The role of liver in the human body is self-evident. Nowadays, liver disease has become a common disease that harms human physical and mental health and is a global high mortality disease. Liver injury is an important factor causing liver disease. Liver injury can be roughly divided into acute liver injury and chronic liver injury. If the formation of liver injury is not controlled, it can further worsen into diseases such as liver fibrosis, liver cirrhosis, and liver cancer. There is no drug with significant therapeutic effect for protecting liver in clinical practice. Therefore, the research on drugs for liver injury is an important topic and has good research prospects.

[0003] Botrychium ternatum (Thunb.) Sw. is a plant of Botrychium ternatum (Thunb.) Sw. and is widely distributed in Shaanxi, Jiangsu, Zhejiang, Fujian, Guangdong and other places in China, and is commonly found in shady and humid shrubs or hillside grasslands at an altitude of 200-2200 meters. The whole plant is used as medicine and is called "a cloud" and "one-legged golden chicken" in traditional Chinese medicine, and has the effects of clearing heat and resolving toxicity, relieving cough and stopping bleeding, and is often used to treat infantile convulsions with high fever, lung heat cough, and sores and toxic swellings. The whole plant of Botrychium ternatum (Thunb.) Sw. mainly contains flavonoids, phenolic acids, polysaccharides, saponins, terpenes, and tannins and other chemical components. Modern pharmacological studies have shown that Botrychium ternatum (Thunb.) Sw. has the effects of antioxidant, antibacterial and anti-inflammatory, antitumor, immune regulation, diuresis and uric acid reduction, and neuroprotection.

[0004] At present, there is still a lack of research on Botrychium ternatum (Thunb.) Sw. in the field of liver function impairment drugs, and its specific effective components, effects and potential mechanisms are still unclear. In view of the serious situation of acute liver injury and the great potential of natural products in this field, in-depth study of the effects and mechanisms of Botrychium ternatum (Thunb.) Sw. in resisting liver injury has important theoretical and practical significance. SUMMARY

[0005] In order to solve the above problems, the present application provides Botrychium ternatum (Thunb.) Sw. and its extract which can prevent or reverse liver injury, and provides an effective drug for treating liver injury, as follows:

[0006] The application of Botrychium ternatum (Thunb.) Sw. and its extract in the preparation of drugs for treating liver function impairment.

[0007] Further, the Botrychium ternatum (Thunb.) Sw. is the dried whole plant of Botrychium ternatum (Thunb.) Sw.

[0008] Further, the extract of the botrychium terrestris is an ethanol extract of the botrychium terrestris, preferably a 70-90% ethanol extract.

[0009] Further, the ethanol extract of the botrychium terrestris is prepared by the following method:

[0010] After the botrychium terrestris is dried and crushed, 500g of the coarse powder is added to 10 times the amount (w / v) of 70-90% ethanol solution (i.e. 500g of medicinal materials is added to 5L of 70% ethanol), and soaked at room temperature for 30min. The extraction is performed twice at 80℃ by hot reflux extraction, each for 2h, and the two extraction solutions are combined. The extraction solution is filtered, and then filtered by a 0.45μm microporous filter membrane. The filtrate is concentrated at 50℃ under reduced pressure until there is no alcohol smell (rotary evaporator pressure: -0.08MPa). The concentrated solution is freeze-dried (-50℃, 48h) to obtain a brown botrychium terrestris ethanol extract freeze-dried powder (BT), which is sealed and stored at -20℃ for standby.

[0011] Further, the drug for treating impaired liver function is a drug for preventing or treating liver damage.

[0012] Further, the drug for treating impaired liver function further comprises one or more pharmaceutically acceptable carriers or excipients. The carriers or excipients include diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. commonly used in the pharmaceutical field. Various dosage forms can be used, such as tablets, powders, injections, granules, powders, capsules or pills, etc.

[0013] A drug for treating impaired liver function comprises botrychium terrestris or an extract thereof. It can be a drug prepared by combining botrychium terrestris or an extract thereof with other drugs, excipients, etc.

[0014] The botrychium terrestris and its extract are used for preventing / treating cell damage and tissue damage caused by oxidation and inflammation.

[0015] Compared with the prior art, the technical effects of the present application are embodied in:

[0016] The present application provides two uses of botrychium terrestris and its extract: (1) for preventing / treating cell damage and tissue damage caused by oxidation and inflammation; and (2) for preventing / treating acute and chronic liver damage. It has been fully verified that the ethanol extract of botrychium terrestris has obvious liver protection effect and dose dependence, and can significantly protect liver damage. It is disclosed in the present application that botrychium terrestris and specific botrychium terrestris extract can also reduce the release of glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST) and alkaline phosphatase (ALP), significantly reduce the degree of liver cell necrosis, and reduce the degree of inflammatory infiltration. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 1 is the effect of Butylthymol Blue (BT) on the survival rate of HepG2 cells induced by paracetamol (APAP).

[0018] Figure 2 Figure 2 is the effect of Butylthymol Blue (BT) on acute liver injury caused by paracetamol (APAP) in mice. DETAILED DESCRIPTION

[0019] The technical solutions of the present application are further limited in combination with specific embodiments, but the scope of protection is not only limited to the description.

[0020] Example 1, Preparation of Butylthymol Blue (BT)

[0021] 1. Experimental content and method.

[0022] Preparation of extract: Take 500g of dried crude powder of Butylthymol Blue (BT), add 10 times the amount (w / v) of 70% ethanol solution (i.e. 500g of medicinal material added with 5L of 70% ethanol), soak at room temperature for 30min. Heat reflux extraction at 80℃ for 2 times, each for 2h, and combine the two extraction solutions. The extraction solution is initially filtered through double-layer gauze, then filtered through a 0.45μm microporous filter membrane, and the filtrate is concentrated at 50℃ under reduced pressure until there is no alcohol taste (rotary evaporator pressure: -0.08MPa). The concentrated solution is freeze-dried (-50℃, 48h) to obtain brownish Butylthymol Blue (BT) freeze-dried powder, which is sealed, stored in the dark at -20℃, and ready for use.

[0023] Chemical composition analysis of BT: The chemical composition of BT was analyzed by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-QE-Orbitrap HRMS). A Hypersil Gold C18 column (2.1 mm x 100 mm, 1.9 μm) was used, with 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B) as the mobile phase, column temperature 35°C, injection volume 2 μL, and flow rate 0.35 mL / min. The gradient elution conditions were as follows: 0-4 min, 2.0%-13.0% B; 4-13 min, 13.0%-36.0% B; 13-14 min, 36.0%-49.0% B; 14-18 min, 49.0%-63.0% B; 18-22 min, 63.0%-98.0% B. An Orbitrap Exploris 240 MS system was used, with a HESI ion source, positive and negative ion mode scanning, a negative ion mode cone hole voltage of 2.5 kV, a positive mode cone hole voltage of 3.5 kV, a desolvation gas temperature of 350°C, an atomization gas pressure of 35 arb, a desolvation gas pressure of 10 arb, a capillary temperature of 320°C, a scanning range m / z 100-1500, an MS resolution of 70000, and an MS / MS resolution of 15000. Finally, MassLynx 4.1 software was used for data collection and analysis.

[0024] 2. Experimental results.

[0025] Example 1, Protective effect of Botrychium ternatum ethanol extract (BT) on acetaminophen-induced HepG2 hepatocyte injury.

[0026] 1. Experimental content and method.

[0027] Preparation of acetaminophen stock solution: 151.16 mg of acetaminophen powder was accurately weighed into 1 mL of cell-grade DMSO, and ultrasonicated to completely dissolve. The stock solution concentration was 1 M, and stored at -20°C. When used, it was diluted to 10 mM with serum-free medium.

[0028] Logarithmic growth phase HepG2 cells were trypsinized and counted (trypan blue staining method verified survival rate ≥ 95%), and cell suspension was prepared at a density of 5 x 10 4 cells / mL. 100 μL of cell suspension (containing 1 x 10 3 cells / well) was inoculated into a 96-well plate, and incubated in a 37°C, 5% CO2 incubator for 24 hours to ensure complete cell adhesion. After discarding the culture medium, 100 μL of drug-containing medium (final drug concentration 10 μM, 3 parallel wells for each concentration, and an equal amount of complete medium without drug was added to the blank control group) was added to each well.

[0029] Blank control group: blank DMEM medium without acetaminophen (added to blank wells without cells);

[0030] Model group: serum-free DME medium pretreated for 3h and then added with 10mM acetaminophen for 24h;

[0031] Positive drug group: serum-free DMEM medium containing 25μg / mL silibinin pretreated for 3h and then added with 10mM acetaminophen for 24h;

[0032] Botrychium ternatum ethanol extract (BT) administration group: serum-free DMEM medium containing 100, 25, 6.25μg / mL of Botrychium ternatum ethanol extract pretreated for 3h and then added with 10mM acetaminophen for 24h;

[0033] The 96-well plate was continuously cultured for 24 hours, and 10μL of CCK-8 reagent was added to each well 30 minutes before the end of the experiment, and incubated in the dark for 1-2 hours. The absorbance (OD value) of each well was measured at 450nm wavelength using a microplate reader. After correction with the blank control group, the cell survival rate was calculated according to the formula (experimental group OD value / blank control OD value) x 100%, and the data was analyzed using GraphPad Prism and a dose-response curve was drawn.

[0034] 2、Experimental results.

[0035] As Figure 1 shown, Botrychium ternatum ethanol extract (BT) showed significant hepatocyte protection effect at a concentration of ≥25μg / mL, with an effect intensity comparable to that of the classic hepatoprotective drug silibinin. It has significant protective activity against acute liver injury and can be used for the preparation of drugs for protecting against acute hepatitis liver injury.

[0036] Example 2, protective effect of Botrychium ternatum ethanol extract (BT) on acute liver injury caused by acetaminophen in mice.

[0037] 1、Materials and methods.

[0038] 6-week-old SPF C57BL male mice were purchased from the Experimental Animal Center of Guizhou Medical University, and were given 12h light and 12h darkness, constant temperature, normal feed and water feeding, and were adapted to the sterile environment for 4 days before the experiment.

[0039] The mice were randomly divided into 6 groups (n=8):

[0040] ① Normal control group: normal saline gavage, no modeling

[0041] ② Model group (APAP group): normal saline gavage + APAP modeling

[0042] 3. Positive drug group (silibinin group): Silibinin 50 mg / kg gavage + APAP modeling

[0043] 4. Low-dose Botrychium ternatum group (50 mg / kg): Botrychium ternatum alcohol extract 50 mg / kg gavage + APAP modeling

[0044] 5. Medium-dose Botrychium ternatum group (100 mg / kg): Botrychium ternatum alcohol extract 100 mg / kg gavage + APAP modeling

[0045] 6. High-dose Botrychium ternatum group (200 mg / kg): Botrychium ternatum alcohol extract 200 mg / kg gavage + APAP modeling

[0046] Drug pretreatment: continuous gavage for 7 days, once a day. According to the mouse 10 mL / kg body weight, Botrychium ternatum alcohol extract (BT) and silybin (Silybin) were dissolved in 0.5% CMC-Na solution. The normal control group and the model group were given the same volume of 0.5% CMC-Na solution.

[0047] APAP liver injury modeling: after the last administration, fasting for 12 h (free drinking water). Intraperitoneal injection of APAP at a dose of 300 mg / kg (dissolved in 40°C preheated normal saline, prepared immediately before use). The normal control group was injected with the same volume of normal saline. Enucleation was performed 24 h after blood collection, and liver tissue was collected after heart perfusion. After standing at room temperature for 2 h, the serum samples were centrifuged at 8000 g for 15 min at 4°C.

[0048] The body weight changes of mice during the entire experiment were recorded. The liver was weighed to record the liver index. Serum samples were subjected to blood biochemical analysis to detect ALT, AST and ALP indicators. After being fixed with paraformaldehyde, the liver tissue was subjected to paraffin embedding, sectioning, HE staining and photographing.

[0049] 2. Experimental results.

[0050] The experimental data of quantitative materials were expressed as mean ± standard deviation. The comparison of two means used one-way ANOVA analysis in SPSS 16.0 statistical software, *p<0.05, **p<0.01, ***p<0.001 compared with the model group.

[0051] Before the experiment, the initial body weight of all mice was measured and recorded. During the entire experimental period, the body weight of mice was measured daily at a fixed time, and the body weight changes were closely observed. As shown in the table, the pre-administration of Silibinin and BT can alleviate the adverse effects of APAP on the body weight of mice to a certain extent, suggesting that they may have a certain protective effect on the body of mice. Figure 2

[0052] ​At the end of the experiment, the relative changes of liver were evaluated by calculating the liver coefficient. As shown in Table 1, it was indicated that Silibinin and BT in middle and high doses had significant protective and improving effects on APAP-induced liver injury in mice. Figure 2

[0053] After the liver cells were damaged, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were released, and the concentrations of these enzymes in serum reflected the degree of liver injury. As shown in Table 2, the blood biochemical results showed that the ethanol extract of Pyrrosia lingii Ching had obvious protective effect and dose dependence, and significantly protected acute liver injury caused by carbon tetrachloride. Figure 2

[0054] The HE staining results showed that the liver cells in the blank control group were arranged in order, the liver lobule structure was clear, and there was no obvious pathological change. The liver tissue in the APAP group showed obvious damage, the liver lobule structure was disordered, and a large area of necrotic area (outlined with dotted line) and abnormal liver cell morphology could be seen. In the Silibinin (50 mg / kg) group, compared with the APAP group, the necrotic area was reduced, but part of the liver tissue was irregular in shape and had certain damage. In the BT (50 mg / kg, 100 mg / kg, 200 mg / kg) group, with the increase of the dose, the necrotic area gradually decreased. In the BT (50 mg / kg) group, obvious necrotic area could be seen; in the 100 mg / kg group, the necrotic area was reduced; in the 200 mg / kg group, the necrotic area was further reduced, the degree of liver tissue damage was reduced, and the structure tended to be normal. In line with the blood biochemical results, it was indicated that the ethanol extract of Pyrrosia lingii Ching had obvious protective effect on liver injury.

[0055] Finally, it should be pointed out that the above examples are only more representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, but can also have many variations. All variations that can be directly derived or inferred by those skilled in the art from the content disclosed in the present application should be considered as falling within the protection scope of the present application.​​

Claims

1. Application of *Pteris vittata* and its extracts in the preparation of drugs for treating liver dysfunction.

2. The application according to claim 1, characterized in that, The aforementioned *Dryopteris crassirhizoma* is the dried whole herb of *Dryopteris crassirhizoma*, a plant belonging to the family Dryopterisceae.

3. The application according to claim 1, characterized in that, The extract of *Dryopteris crassirhizoma* is an ethanol extract of *Dryopteris crassirhizoma*.

4. The application according to claim 1, characterized in that, The ethanol extract of the shady fern is a 70% ethanol extract.

5. The application according to claim 4, characterized in that, The 70% ethanol extract of the *Dryopteris crassirhizoma* was prepared by the following method: After drying and pulverizing *Dryopteris oryzae*, 500g of the dried coarse powder was added to 10 times the volume (w / v) of 70% ethanol solution and soaked at room temperature for 30min. The mixture was then extracted twice by hot reflux at 80℃ for 2h each time, and the two extracts were combined. After initial filtration, the extract was filtered through a 0.45μm microporous membrane, and the filtrate was concentrated under reduced pressure at 50℃ until no alcohol odor remained. The extract was then freeze-dried (-50℃, 48h) to obtain a brownish-brown freeze-dried powder of *Dryopteris oryzae* ethanol extract, which was sealed and protected from light at -20℃ for later use.

6. The application according to claim 1, characterized in that, The aforementioned medication for treating liver dysfunction is a drug used to prevent or treat liver damage.

7. The application according to claim 1, characterized in that, The aforementioned medication for treating liver dysfunction also contains pharmaceutically acceptable carriers or excipients.

8. A drug for treating liver dysfunction, characterized in that, It contains *Dryopteris crassirhizoma* or its extracts.

9. The medicament for treating liver dysfunction according to claim 7, characterized in that, The aforementioned drug for treating liver dysfunction is prepared by combining *Dryopteris crassirhizoma* or its extract with other drugs, excipients, etc.

10. Application of *Pteris vittata* and its extracts in the preparation of drugs for the prevention / treatment of cell and tissue damage caused by oxidation and inflammation.

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