Application of methyl American tea alkane in preparation of medicine with anti-hepatic fibrosis effect

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to application of methyl American tea alkane in preparation of a medicine with an anti-hepatic fibrosis effect. Researches show that the methyl American tea alkane has an anti-hepatic fibrosis effect; in addition, the methyl American tea alkane can also significantly reduce the expression of liver fibrosis markers such as alpha-SMA and Col-I; and the compound also has an excellent effect of resisting TGF-beta1 induced hepatic fibrosis and an effect of inhibiting hepatic stellate cell proliferation or promoting hepatic stellate cell apoptosis. Therefore, the methyl American tea alkane serving as an active ingredient is used for preparing the medicine with the anti-hepatic fibrosis effect, especially the medicine with the anti-TGF-beta1 induced hepatic fibrosis effect or the medicine with the hepatic stellate cell proliferation inhibiting effect or the hepatic stellate cell apoptosis promoting effect, and the methyl American tea alkane has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, and in particular to application of methyl tantalum in preparing a drug with an anti-liver fibrosis effect. Background Art

[0002] Liver fibrosis is the most common pathological feature of most chronic liver diseases. It is mainly manifested as excessive deposition of extracellular matrix (especially collagen) in the liver. It is characterized by a large amount of extracellular matrix accumulation. The damage cannot be restored in time, resulting in the gradual replacement of liver parenchyma by fibrotic tissue. Its continuous deterioration gradually develops into cirrhosis and eventually liver cancer. Cirrhosis and liver cancer will bring considerable economic burden to society and are one of the public health issues that seriously threaten people's health. Epidemiological data show that cirrhosis affects hundreds of millions of people worldwide. It ranks 14th among the common causes of death among adults worldwide (cirrhosis causes about 1.03 million deaths each year). Therefore, actively exploring the pathogenesis of liver fibrosis and seeking effective treatment measures are of great significance to reducing the incidence and mortality of cirrhosis.

[0003] The activation of hepatic stellate cells (HSCs) is the main process of liver fibrosis. At the same time, the activated HSCs can further transform into myofibroblasts, which is directly related to the formation of extracellular matrix (ECM). When the proliferation of HSCs is enhanced, ECM is overproduced and accumulated in the liver, the composition of ECM changes from type IV and type VI collagen, glycoproteins and proteoglycans to type I and type III collagen and fibronectin, causing liver fibrosis. Studies have shown that this pathological process is reversible, so regulating HSCs can have a therapeutic effect on liver fibrosis.

[0004] At present, there are many drugs used to treat liver fibrosis. However, Western medicine often only targets a single gene or protein and is prone to cause many adverse reactions. Traditional Chinese medicine has the advantages of reliable efficacy and low toxicity and side effects for many chronic diseases and has been valued by the medical community. Although the mechanism of action and active ingredients of most traditional Chinese medicines are unclear and there is a lack of unified diagnostic and treatment standards, they have the characteristics of diverse structures, low toxicity, and a wide range of sources, and have unique advantages and great potential in the fight against liver fibrosis.

[0005] Triterpenoids are a class of compounds derived from methylvaleric acid and having a basic carbon frame of 6 isoprenoid structural units. Triterpenoids are widely present in various plant organisms in nature. Some common Chinese herbal medicines, such as ginseng, Panax notoginseng and licorice, all contain triterpenoids and have good biological activity. The triterpenoids reported in Hovenia dulcis are commonly found in Hovenia dulcis plants Hovenia dulcis and Hovenia dulcis, mainly in the form of dammarane, lupine, American tea type, olean type and chain triterpenoids. The methyl American tea involved in the present invention belongs to the American tea type. According to existing reports (Yang, J., et al., Hovendulcisic acid AD: four novel ceanothane-type triterpenoids from Hovenia dulcis stems with anticancer properties. Front Chem, 2024.12: p.1383886.), the A and E rings of the ceanothane-type structure undergo isomerization at the same time, and the substituents at C-1, 27, and 28 change [1]. The methyl ceanothane molecular formula C 31 H 48 O5, molecular weight is 500.71, and its structural formula is shown in Ⅰ:

[0006]

[0007] However, the prior art does not report that methyl ester of tert-butyl ester has an anti-hepatic fibrosis effect. Summary of the invention

[0008] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides the use of methyl teatane in preparing a drug having an anti-liver fibrosis effect.

[0009] The technical solution of the present invention is as follows:

[0010] The present invention firstly provides an application of methyl teatane in preparing a medicine with an anti-liver fibrosis effect.

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

[0012] Preferably, the anti-liver fibrosis effect is exerted by inhibiting the expression of α-SMA and Col-I protein.

[0013] Preferably, the anti-liver fibrosis effect is achieved by improving the ability to resist oxidative stress.

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

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

[0016] The present invention also provides a use of the methyl teacaine in preparing a medicine having the function of inhibiting the proliferation of hepatic stellate cells or promoting the apoptosis of hepatic stellate cells.

[0017] Preferably, the drug contains methyl teatane as an active ingredient and further comprises pharmaceutically acceptable excipients.

[0018] Preferably, the weight content of methyl ester in the medicine is 0.1-99.9%.

[0019] Preferably, the drug is prepared into powder, pill, tablet, capsule, oral solution, aerosol or injection.

[0020] The present invention also provides a composition or extract comprising methyl-tetracycline.

[0021] The present invention also provides a use of the above composition or extract in preparing a medicine with anti-liver fibrosis effect.

[0022] Preferably, the drug for anti-liver fibrosis is a drug for anti-TGF-β1-induced liver fibrosis.

[0023] The present invention also provides an application of the above composition or extract in preparing a medicine having the function of inhibiting the proliferation of hepatic stellate cells or promoting the apoptosis of hepatic stellate cells.

[0024] Beneficial effects: The present invention provides a new use of methyl oleic acid tea. Studies have shown that the methyl oleic acid tea has an anti-hepatic fibrosis effect. In addition, the methyl oleic acid tea can significantly reduce the expression of liver fibrosis markers such as α-SMA and Col-I. It also has excellent anti-TGF-β1-induced liver fibrosis effect and inhibits hepatic stellate cell proliferation or promotes hepatic stellate cell apoptosis. Therefore, using methyl oleic acid tea as an active ingredient to prepare a drug with an anti-hepatic fibrosis effect, especially a drug with an anti-TGF-β1-induced liver fibrosis effect or a drug with an inhibitory effect on hepatic stellate cell proliferation or apoptosis of hepatic stellate cells has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of four American tea type triterpenes 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 the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0026] Figure 2 This is the effect of methyl tea alcohol on the 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 the model group, # P<0.05, ## P<0.01, ### P<0.001.

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

[0028] Figure 4 The effect of methyl tea alcohol 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 the model group, # P<0.05, ## P<0.01. ### P<0.001.

[0029] Figure 5 The effect of methyl tea alcohol on the antioxidant effect of HSC-T6 cells; in the figure, compared with the blank group, * P<0.05, ** P < 0.01; ** P<0.001; compared with the model group, # P<0.05, ## P<0.01. ### P<0.001. DETAILED DESCRIPTION

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

[0031] Example 1 Inhibitory effects of different American tea type triterpenes on proliferation and activation of HSC-T6 cells

[0032] HSC-T6 cell culture: The cells were inoculated in DMEM (containing penicillin + streptomycin) medium containing 10% FBS and cultured in a 37°C, 5% CO2 incubator. When the cells were in the logarithmic growth phase, they were digested and passaged with trypsin containing EDTA. The cells were seeded in a 96-well plate with 5,000 cells per well; they were 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 with a medium containing 0.7% FBS for 24 h.

[0033] CCK8 method to detect HSC-T6 cell proliferation: After 24 hours of action in each group, the culture medium in the wells was aspirated, washed twice with PBS, 10 μL CCK8 was added to each well, mixed and incubated in the incubator for 2 hours, and the absorbance (A) value of each well at a wavelength of 450 nm was detected by an enzyme-labeled instrument. The OD value of the sample to be tested and the blank control at 450 nm was read by the enzyme-labeled instrument, and the cell survival rate of HSC at different drug concentrations was calculated. Cell survival rate = [(experimental well - blank well) / (control well - blank well)] × 100%

[0034] The experimental results are as follows:

[0035] The experimental results are as follows Figure 1 As shown by Figure 1 It can be seen that the four types of acetyl tea triterpenes have a certain inhibitory effect on the proliferation of HSC-T6 cells; among them, the methyl acetyl tea (Methylceanothate) described in the present invention has the most obvious dose-effect relationship. Methyl acetyl tea at a concentration of 10 μM began to significantly inhibit the proliferation of HSC-T6 cells (P < 0.001). The results of this experimental example show that the mechanism of methyl acetyl tea in exerting anti-liver fibrosis may be to inhibit the proliferation of hepatic stellate cells, and the anti-liver fibrosis effect of methyl acetyl tea is far superior to other types of acetyl tea triterpenes.

[0036] Example 2 Toxicity of Methyl-Tetracarboxylic Acid to L-02 and AML-12 Cells

[0037] L-02 / AML-2 cell culture: The cells were inoculated in RPMI-1640 (containing penicillin + streptomycin) medium containing 10% FBS and cultured in a 37°C, 5% CO2 incubator. When the cells were in the logarithmic growth phase, they were digested and passaged with trypsin containing EDTA. The cells were seeded in a 96-well plate with 5,000 cells per well; they were divided into a normal group and a methyl-americane (2.5, 5, 10, 20, 40, 80, 160, 320, 640 μM) group, and cultured with a medium containing 10% FBS for 24 h.

[0038] CCK8 method to detect L-02 cell proliferation: After 24 hours of action in each group, the culture medium in the wells was aspirated, washed twice with PBS, 10 μL CCK8 was added to each well, mixed and incubated in the incubator for 2 hours, and the absorbance (A) value of each well at a wavelength of 450 nm was detected by an enzyme-labeled instrument. The OD value of the sample to be tested and the blank control at 450 nm was read by the enzyme-labeled instrument, and the cell survival rate of HSC at different drug concentrations was calculated. Cell survival rate = [(experimental well - blank well) / (control well - blank well)] × 100%

[0039] The experimental results are as follows:

[0040] The experimental results are as follows Figure 2 As shown by Figure 2 It can be seen that methyl oleic acid has an inhibitory effect on the proliferation of L-02 and AML-12 cells when the concentration used exceeds 80 μM. The results of this experimental example show that methyl oleic acid inhibits the proliferation of hepatic stellate cells (HSC-T6) and has a small toxic effect on normal cells (L-02 and AML-12).

[0041] Example 3 Effect of Methyl American Tea on the Expression of Fibrosis Marker Proteins and Oxidative Stress Marker Proteins in HSC-T6 Cells

[0042] Experimental method: Take cells from each group after 24 hours of treatment, scrape total protein using RIPA protein lysis buffer method, and quantify protein using BCA method. Dissolve equal amounts of protein in sodium dodecyl sulfate (SDS) sample buffer, separate by 10% SDS-polyacrylamide gel electrophoresis, and transfer to PVDF membrane. Then add primary antibodies α-SMA and COL-I, and incubate overnight at 4°C. Wash three times with TBST at room temperature, 10 minutes each time. Then incubate with secondary rabbit anti-IgG for 1 hour, and wash the membrane three times, 5 minutes each time. Then infiltrate the PVDF membrane with ECL and scan it under a chemiluminescence imaging system. Use GAPDH protein as an internal reference.

[0043] The experimental results are as follows:

[0044] TGF-β1 is a type of polypeptide that regulates cell growth and differentiation, can promote collagen production, activates HSC, promotes liver collagen gene expression, and promotes ECM synthesis. TGF-β1 is the strongest factor promoting liver fibrosis. α-SMA protein is mainly present in smooth muscle and myofibroblasts. When liver fibrosis occurs in liver tissue, activated HSCs will express α-SMA, COL-I and COL-Ⅲ. Therefore, α-SMA, COL-I and COL-Ⅲ can be regarded as markers of HSC activation.

[0045] The experimental results are shown in Figure 3 ,Depend on Figure 3 It can be seen that methyl tea alkane 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 experimental example show that methyl tea alkane inhibits the activation of HSCs and reduces the expression of activation marker proteins α-SMA, COL-I and COL-Ⅲ to play an anti-liver oxidative effect.

[0046] Example 4 Effect of Methyl-Tetradecane on Apoptosis of HSC-T6 Cells

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

[0048] The experimental results are as follows:

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

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

[0051] Example 5 Antioxidant effect of methyl-tetracarboxylic acid on HSC-T6 cells

[0052] Experimental method: HSC-T6 cells in the logarithmic growth phase were taken and inoculated in a 6-well plate at 150,000 / well, with three replicate wells in each group, and placed in a cell culture incubator overnight; the culture medium was discarded, and drug treatment was performed according to the control group, model group and drug treatment group, and culture was continued for 24 hours; after the culture was completed, the culture medium was discarded and PBS was washed twice; 1 mL of DCFH-DA dilution was added to each well and incubated in an incubator for 20 minutes; after washing three times with serum-free medium, the plates were observed and photographed 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 liver cells. When the liver is damaged, HSCs are activated by TGF-β1 and secrete a large number of inflammatory factors. The damaged liver cells will produce excessive ROS, which will cause an imbalance in the ratio of MMPs and TIMPs, leading to liver fibrosis. Therefore, the anti-liver fibrosis effect can be achieved by improving the ability to resist oxidative stress.

[0055] The experimental results are shown in Figure 3 and Figure 5 . Figure 3 The results showed that compared with the blank group, the TGF-β1 model group could significantly reduce the content of HO-1 and NQO1 (HO-1 and NQO1 are important indicators of anti-oxidative stress). After the administration of methyl tantalum, the expression levels of HO-1 and NQO1 began to increase. Figure 5 The results of ROS fluorescence showed that compared with the blank group, the TGF-β1 model group could significantly increase the content of ROS, and after the administration of methyl oleic acid, the ROS index began to decrease. This suggests that methyl oleic acid has antioxidant ability, and its anti-liver fibrosis mechanism may be related to its anti-oxidative stress ability.

Claims

1. The application of methyl tealane in the preparation of drugs with anti-liver fibrosis effect.

2. The use according to claim 1, characterized in that: The anti-liver fibrosis is anti-TGF-β1-induced liver fibrosis. Preferably, the anti-liver fibrosis effect refers to promoting TGF-β1-induced hepatic stellate cell apoptosis to exert the anti-liver fibrosis effect.

3. The use of methyl tealane in the preparation of drugs that inhibit the proliferation of hepatic stellate cells or promote the apoptosis of hepatic stellate cells.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine uses methyl teatane as an active ingredient and also contains pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The weight content of methyl tantalum in the medicine is 0.1-99.9%.

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

7. A composition or extract, characterized in that Contains Methyl Amyltane.

8. Use of the composition or extract according to claim 7 in the preparation of a drug having an anti-liver fibrosis effect.

9. The use according to claim 8, characterized in that: The drug for resisting liver fibrosis is a drug for resisting liver fibrosis induced by TGF-β1.

10. Use of the composition or extract according to claim 7 in the preparation of a medicament having the effect of inhibiting the proliferation of hepatic stellate cells or promoting the apoptosis of hepatic stellate cells.

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