Use of methyl clemoplatine in the preparation of a drug with anti-hepatic fibrosis effect

Abstract

The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of methyl american teaflavane in preparation of a medicine with an anti-hepatic fibrosis effect.Researches show that the methyl american teaflavane has an anti-hepatic fibrosis effect; in addition, the methyl american teaflavane can also significantly reduce expression of hepatic fibrosis markers such as alpha-SMA and Col-I; and has an excellent anti-TGF-beta 1 induced hepatic fibrosis effect and an inhibiting hepatic stellate cell proliferation or promoting hepatic stellate cell apoptosis effect.Therefore, the methyl american teaflavane is used as an active ingredient to prepare a medicine with an anti-hepatic fibrosis effect, especially a medicine with an anti-TGF-beta 1 induced hepatic fibrosis effect or a medicine with an inhibiting hepatic stellate cell proliferation or promoting hepatic stellate cell apoptosis effect, which has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of methyl american teaflavane in preparation of a medicine with an anti-hepatic fibrosis effect. BACKGROUND

[0002] Hepatic fibrosis is the most common pathological feature of most chronic liver diseases, mainly manifested as diffuse extracellular matrix (especially collagen) over-deposition in the liver, characterized by massive accumulation of extracellular matrix, damage that cannot be timely recovered, and replacement of liver parenchyma by fibrotic tissue, which gradually develops into cirrhosis and eventually develops into liver cancer. Liver cirrhosis and liver cancer have brought considerable economic burden to the society, and are one of the public health problems that seriously threaten the health of the public. Epidemiological data shows that liver cirrhosis affects billions of people worldwide, and it is the 14th common cause of death among adults in the world (liver cirrhosis causes about 1.03 million deaths per year). Therefore, actively exploring the pathogenesis of hepatic fibrosis and seeking effective treatment measures have important significance for reducing the incidence and mortality of liver cirrhosis.

[0003] Activation of hepatic stellate cells (HSCs) is the main process of hepatic fibrosis, and activated hepatic stellate cells can further transform into myofibroblasts, which are directly related to the formation of extracellular matrix (ECM). When the proliferation of hepatic stellate cells is enhanced, ECM is excessively produced and accumulated in the liver, and the composition of ECM is transformed from type IV and type VI collagen, glycoprotein and proteoglycan to type I and type III collagen and fibronectin, causing hepatic fibrosis. Studies have shown that this pathological process is reversible, so regulating HSCs can have a therapeutic effect on hepatic fibrosis.

[0004] At present, there are many drugs for treating hepatic fibrosis. However, western medicines often only target a single gene or protein, and are prone to cause many adverse reactions. Traditional Chinese medicines have the advantages of reliable curative effect, small toxicity and side effects, and are valued by the medical field. Although the mechanism of action and effective components of most traditional Chinese medicines are not clear, and there is a lack of unified diagnosis and treatment standards, they have the characteristics of structural diversity, low toxicity, and wide sources, and have unique advantages and great potential in the treatment of hepatic fibrosis.

[0005] Triterpenoids are a class of compounds derived from ketal, with a basic carbon skeleton of six isoprene structural units. Triterpenoids are widely found in various plant-based organisms in nature. Some common Chinese herbal medicines, such as ginseng, sanchi and licorice, contain triterpenoids and have good biological activity. The triterpenoids reported in this paper are commonly found in Hovenia dulcis and Hovenia acerba. They are mainly dammarane type, lupine type, ceanothane type, oleanane type and chain triterpenoids. The methyl ceanothane involved in this invention belongs to the ceanothane type. According to the existing reports (Yang, J., et al., Hovendulcisic acid A-D: four novel ceanothane-typetriterpenoids from Hovenia dulcis stems with anticancer properties. Front Chem, 2024. 12: p. 1383886.), the A ring and E ring of the ceanothane type structure undergo isomerization, and the C-1, 27, 28 substitution group changes [1]. The molecular formula of the methyl ceanothane is C 31 H 48 O5, with a molecular weight of 500.71, and its structural formula is shown as I:

[0006]

[0007] However, the prior art does not report that methyl ceanothane has an anti-hepatic fibrosis effect. SUMMARY

[0008] In order to overcome at least one of the technical problems in the prior art, the present application provides the use of methyl ceanothane in the preparation of a drug with an anti-hepatic fibrosis effect.

[0009] The technical scheme of the present application is as follows:

[0010] The present application first provides the use of methyl ceanothane in the preparation of a drug with an anti-hepatic fibrosis effect.

[0011] Preferably, the anti-hepatic fibrosis effect is achieved by promoting the apoptosis of hepatic stellate cells.

[0012] Preferably, the anti-hepatic fibrosis effect is achieved by inhibiting the expression of alpha-SMA and Col-I protein.

[0013] Preferably, the anti-hepatic fibrosis effect is achieved by improving the antioxidant stress capacity.

[0014] Preferably, the anti-hepatic fibrosis is anti-TGF-β1-induced hepatic fibrosis.

[0015] Preferably, the anti-hepatic fibrosis refers to the anti-hepatic fibrosis effect by promoting TGF-β1-induced hepatic stellate cell apoptosis.

[0016] The present application also provides a use of the above-mentioned methylamericantiaflavan in the preparation of a medicament with the effect of inhibiting hepatic stellate cell proliferation or promoting hepatic stellate cell apoptosis.

[0017] Preferably, the medicament with methylamericantiaflavan as the active ingredient further comprises a pharmaceutically acceptable excipient.

[0018] Preferably, the content of methylamericantiaflavan in the medicament is 0.1-99.9%.

[0019] Preferably, the medicament is prepared into a powder, a pill, a tablet, a capsule, an oral liquid, an aerosol or an injection.

[0020] The present application also provides a composition or extract comprising methylamericantiaflavan.

[0021] The present application also provides a use of the above-mentioned composition or extract in the preparation of a medicament with the effect of anti-hepatic fibrosis.

[0022] Preferably, the medicament with the effect of anti-hepatic fibrosis is a medicament with the effect of anti-TGF-β1-induced hepatic fibrosis.

[0023] The present application also provides a use of the above-mentioned composition or extract in the preparation of a medicament with the effect of inhibiting hepatic stellate cell proliferation or promoting hepatic stellate cell apoptosis.

[0024] Beneficial effects: the present application provides a brand-new use of methylamericantiaflavan; researches show that the methylamericantiaflavan has the effect of anti-hepatic fibrosis; in addition, the methylamericantiaflavan can significantly reduce the expression of liver fibrosis markers such as α-SMA and Col-I; and has excellent effects of anti-TGF-β1-induced hepatic fibrosis, inhibiting hepatic stellate cell proliferation or promoting hepatic stellate cell apoptosis. Therefore, the methylamericantiaflavan is used as the active ingredient to prepare a medicament with the effect of anti-hepatic fibrosis, especially a medicament with the effect of anti-TGF-β1-induced hepatic fibrosis or a medicament with the effect of inhibiting hepatic stellate cell proliferation or promoting hepatic stellate cell apoptosis has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Effects of four kinds of triterpenes of the methylamericantiaflavan type on the proliferation of HSC-T6 cells; in the figure, compared with the blank group, * P<0.05, **P<0.01; ** P<0.001; compared with model group, # P<0.05, ## P<0.01, ### P<0.001.

[0026] Figure 2 Effects of methylamericantiaxanthine on proliferation of L-02 and AML-12 cells; in the figure, compared with the blank group, * P<0.05, ** P<0.01; ** P<0.001; compared with model group, # P<0.05, ## P<0.01, ### P<0.001.

[0027] Figure 3 Effects of methylamericantiaxanthine on expression of α-SMA, Col-I, Col-III, HO-1 and NQO1 proteins of HSC-T6 cells; in the figure, compared with the blank group, * P<0.05, ** P<0.01; ** P<0.001; compared with model group, # P<0.05, ## P<0.01, ### P<0.001.

[0028] Figure 4 Effects of methylamericantiaxanthine on apoptosis of HSC-T6 cells; in the figure, compared with the blank group, * P<0.05, ** P<0.01; ** P<0.001; compared with model group, # P<0.05, ## P<0.01. ### P<0.001.

[0029] Figure 5 Effects of methylamericantiaxanthine on antioxidation of HSC-T6 cells; in the figure, compared with the blank group, * P<0.05, ** P<0.01; ** P<0.001; compared with model group, # P<0.05, ## P<0.01. ### P<0.001. DETAILED DESCRIPTION

[0030] The present application is further explained in conjunction with specific examples, which do not limit the present application in any form.

[0031] Example 1 Inhibitory effects of different ceanothate triterpenes on the proliferation and activation of HSC-T6 cells

[0032] Culture of HSC-T6 cells: The cells were inoculated in DMEM medium containing 10% FBS and penicillin + streptomycin, and cultured in a 37°C, 5% CO2 incubator. When the cells were in the logarithmic growth phase, they were passaged by trypsin digestion with EDTA. The cells were seeded in a 96-well plate at 5000 cells per well, and divided into a normal group, a TGF-β1 (10 ng / mL) group, and a TGF-β1 (10 ng / mL) + drug (2.5, 5, 10, 20, 40, 80, 90, 100 μM) group, and cultured in a medium containing 0.7% FBS for 24 h.

[0033] CCK8 method for detecting the proliferation of HSC-T6 cells: The culture medium in each well was aspirated after 24 h of action, and the cells were washed twice with PBS. 10 μL of CCK8 was added to each well, mixed well, and incubated in an incubator for 2 h. The absorbance (A) value of each well at 450 nm was detected by an enzyme marker. The OD value of the test sample and the blank control at 450 nm was read by an enzyme marker, and the cell survival rate of HSC under different drug concentrations was calculated. Cell survival rate = [(experimental well - blank well) / (control well - blank well)] x 100%

[0034] The experimental results are as follows:

[0035] The experimental results are as shown in Figure 1 It can be seen from Figure 1 that the four ceanothate triterpenes have a certain inhibitory effect on the proliferation of HSC-T6 cells. Among them, methylceanothate has the most obvious dose-effect relationship. Methylceanothate at a concentration of 10 μM significantly inhibited the proliferation of HSC-T6 cells (P < 0.001). The results of this experiment show that the mechanism of methylceanothate in preventing liver fibrosis may be to inhibit the proliferation of hepatic stellate cells, and the effect of methylceanothate in preventing liver fibrosis is much better than that of other types of ceanothate triterpenes.

[0036] Example 2 Toxic effects of methylceanothate on L-02 and AML-12 cells

[0037] L-02 / AML-2 cell culture: Cells were seeded in RPMI-1640 medium (containing penicillin and streptomycin) with 10% FBS and cultured at 37°C in a 5% CO2 incubator. When the cells were in the logarithmic growth phase, they were passaged using trypsin containing EDTA. Cells were seeded into 96-well plates at 5000 cells per well; divided into a normal group and a methyl thiazoline (2.5, 5, 10, 20, 40, 80, 160, 320, 640 μM) group, and cultured in medium containing 10% FBS for 24 h.

[0038] CCK8 assay for L-02 cell proliferation: After 24 hours of treatment, the culture medium in each well was aspirated, and the cells were washed twice with PBS. 10 μL of CCK8 was added to each well, mixed thoroughly, and incubated for 2 hours. The absorbance (A) value of each well at 450 nm was measured using a microplate reader. The OD values ​​of the test samples and blank controls at 450 nm were read using the microplate reader, and the cell viability of HSCs at different drug concentrations was calculated. Cell viability = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%

[0039] The experimental results are as follows:

[0040] Experimental results are as follows Figure 2 As shown, by Figure 2 It was found that methyl meropenem inhibited the proliferation of L-02 and AML-12 cells at concentrations exceeding 80 μM. The results of this experiment indicate that methyl meropenem inhibits the proliferation of hepatic stellate cells (HSC-T6) while exhibiting minimal toxicity to normal cells (L-02 and AML-12).

[0041] Example 3: Effects of methyltheanine on the expression of fibrosis marker proteins and oxidative stress marker proteins in HSC-T6 cells

[0042] Experimental Methods: Cells from each group were treated for 24 hours. Total protein was collected using the RIPA protein lysis buffer method and quantified using the BCA method. Equal volumes of protein were dissolved in sodium dodecyl sulfate (SDS) sample buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. Primary antibodies against α-SMA and COL-I were added, and the membrane was incubated overnight at 4°C. The membrane was washed three times with TBST at room temperature for 10 minutes each time. Then, it was incubated with rabbit anti-IgG secondary antibody for 1 hour, followed by three more washes for 5 minutes each. The PVDF membrane was then infiltrated with ECL and scanned using a chemiluminescence imaging system. GAPDH protein was used as an internal control.

[0043] The experimental results are as follows:

[0044] TGF-β1 is a kind of polypeptide that regulates cell growth and differentiation, can promote the generation of collagen, has the effect of activating HSC, promoting the expression of collagen gene in liver, promoting the synthesis of ECM, and TGF-β1 is the strongest promoting factor of liver fibrosis. The α-SMA protein mainly exists in smooth muscle and myofibroblasts, and when liver fibrosis occurs in liver tissue, activated HSCs will express α-SMA, COL-I and COL-III, so α-SMA and COL-I and COL-III can be regarded as markers of HSC activation.

[0045] The experimental results are shown in Figure 3 It can be seen from Figure 3 that methyl american teaflavone has an inhibitory effect on the expression of α-SMA, COL-I and COL-III proteins in HSC-T6 induced by TGF-β1, and has a significant dose-effect relationship. The results of this experiment show that methyl american teaflavone inhibits the activation of HSCs, reduces the expression of activated marker proteins α-SMA, COL-I and COL-III, and plays an anti-liver fibrosis role.

[0046] Example 4 Effect of methyl american teaflavone on HSC-T6 cell apoptosis

[0047] Experimental method: HSC-T6 cells were taken, treated with drugs for 24 h, then washed with phosphate buffered saline PBS twice, then trypsin digestion, collected cells, centrifuged, discarded the supernatant to prepare cell suspension, added 5 μL of Annexin V-FITC staining solution, mixed, then added 10 μL of propidium iodide staining, mixed, 37℃ for 4 min, PI staining, then detected the apoptosis of cells in each group by flow cytometry.

[0048] The experimental results are as follows:

[0049] During the process of liver fibrosis, the differentiation of hepatic stellate cells (HSCs) into proliferative myofibroblasts is the core process of liver fibrosis. Promoting the apoptosis of HSCs and reducing the number of activated HSCs is a key link to reverse or delay liver fibrosis.

[0050] The experimental results are shown in Figure 4 , Figure 4 The flow cytometry and fluorescence results show that methyl american teaflavone at a concentration of 8 μM can significantly inhibit the apoptosis of HSC-T6 cells (P<0.05). Methyl american teaflavone plays an anti-liver fibrosis role by promoting the apoptosis of TGF-β1 induced HSC-T6 cells. This experiment shows that methyl american teaflavone may promote the apoptosis of activated HSCs, inhibit the expression of α-SMA and COL-I, and inhibit the occurrence of liver fibrosis.

[0051] Example 5 Antioxidant effect of methyl american teaflavone on HSC-T6 cells

[0052] Experimental method: Take the logarithmic growth phase of HSC-T6 cells, 150,000 cells per hole are inoculated in 6-hole plates, three replicate holes are set in each group, and placed in the cell culture box overnight; discard the culture medium, and perform drug treatment according to the control group, model group and drug group, and continue to culture for 24h; after the culture is finished, the culture medium is discarded, and PBS is washed twice; add 1mL of DCFH-DA diluent to each hole, and incubate in the incubator for 20min; after washing with serum-free medium for three times, observe and take pictures under an inverted fluorescence microscope.

[0053] The experimental results are as follows:

[0054] There are a large number of organelles and enzymes that can produce ROS in hepatocytes, when liver injury occurs, HSCs are activated by TGF-β1, and a large number of inflammatory factors are secreted, damaged hepatocytes will produce excess ROS, which will cause the imbalance of the ratio of MMPs and TIMPs, leading to liver fibrosis. Therefore, the anti-oxidative stress capacity can be improved to achieve the effect of anti-liver fibrosis.

[0055] The experimental results are as follows: Figure 3 and Figure 5 . Figure 3 The results show that compared with the blank group, the TGF-β1 model group can significantly reduce the content of HO-1 and NQO1 (HO-1 and NQO1 are important indicators of antioxidant stress), and after giving methyl american teaflavane, the expression of HO-1 and NQO1 begins to increase. Figure 5 The ROS fluorescence results show that compared with the blank group, the TGF-β1 model group can significantly increase the content of ROS, and after giving methyl american teaflavane, the ROS index begins to decrease. It is suggested that methyl american teaflavane has antioxidant capacity, and its anti-liver fibrosis mechanism may be related to the antioxidant stress capacity.

Claims

1. The use of a compound as the sole active ingredient in the manufacture of a medicament having an anti-hepatic fibrosis effect, characterized in that, The structural formula of the compound is shown as formula I. Formula I.

2. Use according to claim 1, characterized in that, The anti-hepatic fibrosis is anti-TGF- β 1 induced hepatic fibrosis.

3. Use according to claim 2, characterized in that, The anti-hepatic fibrosis refers to playing an anti-hepatic fibrosis role by promoting TGF-β1 induced hepatic stellate cell apoptosis.

4. The use according to any one of claims 1 to 3, characterized in that, The medicine also comprises a pharmaceutically acceptable auxiliary.

5. Use according to claim 4, characterized in that, The weight content of the compound with the structure shown as formula I in the medicine is 0.1-99.9%.

6. Use according to claim 4, characterized in that, The medicine is prepared into a powder, a pill, a tablet, a capsule, an oral liquid, an aerosol or an injection.

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