Application of N1, N5, N10-tri-p-coumaroyl spermidine in preparation of liver drugs

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of N1, N5, N10-tri-p-coumaroyl spermidine in the aspect of preparing liver medicines. The invention discloses the application of the N1, N5 and N10-three pairs of coumaroyl spermidine (the chemical name is N1, N5, N10-tri-p-coumaroyl spermidine, the molecular formula is C34H37N3O6, the molecular weight is 583.7, and hereinafter referred to as 3NS) in preparation of the medicine for preventing and treating hepatic fibrosis and hepatic injury for the first time. Compared with the prior art, the compound shows low toxicity in hepatic fibrosis and hepatic injury, can remarkably inhibit abnormal increase of fibrosis markers COL1A1, COL1A2, FN1 and CTGF in hepatic stellate cells LX-2 induced by TGF-beta1, meanwhile, improves the survival rate of AML-12 hepatic cells damaged by acetaminophen induction, and the increase shows concentration dependence, so that the compound has good effects of preventing and treating hepatic fibrosis and hepatic injury, and can be used for preparing the medicine for treating hepatic fibrosis and hepatic injury. And the traditional Chinese medicine composition has a certain hepatocyte proliferation promoting effect. The 3NS not only has a good effect of preventing and treating hepatic fibrosis, but also has the effects of promoting liver cell proliferation and protecting the liver from liver injury caused by chemical substances, so that the 3NS is suitable for preparing medicines for treating liver-related diseases such as hepatic fibrosis and liver injury caused by chemical substances or medicines.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and specifically relates to N 1 ,N 5 ,N 10 -Application of tri-p-coumaryl spermidine in the preparation of liver drugs. Background Art

[0002] The liver is the largest substantial organ in the human body, and undertakes key functions such as metabolism, immune regulation, and detoxification. Liver damage caused by viral hepatitis, alcohol and drug toxic liver disease, and systemic diseases, as well as the subsequent liver fibrosis, cirrhosis, and liver failure, have become common diseases that seriously endanger human health. Liver fibrosis is a repair response to chronic liver damage caused by long-term and repeated stimulation of the liver by various pathogenic factors, resulting in excessive or abnormal deposition of extracellular matrix. Liver fibrosis is mainly characterized by liver cell damage, which activates hepatic stellate cells through paracrine effects, causing them to synthesize and secrete a large amount of type I collagen, fibronectin and other components, and causing a large amount of extracellular matrix (ECM) to accumulate in the liver. Inhibiting the synthesis and secretion of fibrosis markers such as type I collagen and fibronectin by hepatic stellate cells is an effective means to prevent or even reverse liver fibrosis. Chemical liver injury refers to liver damage induced by various chemical substances (including drugs) and their metabolites, and is one of the main causes of acute liver injury. At present, more than 1,100 drugs known to be marketed worldwide have been found to have potential hepatotoxicity. The most common liver-damaging drugs in China are mainly the antipyretic and analgesic acetaminophen (APAP), anti-tuberculosis drugs (isoniazid, rifampicin), etc.

[0003] At present, there is a lack of specific treatment for chemical liver injury in clinical practice. The principle of treatment is to protect liver function, and prevention remains the focus. Therefore, finding effective drugs to prevent and treat liver fibrosis and liver injury is urgent for the development of liver disease. After the liver is damaged, such as after partial liver resection or massive death of liver cells (such as in drug-induced liver injury or viral infection), the remaining liver cells will proliferate to replenish the lost cells and restore liver function. Therefore, the proliferation of liver cells helps the liver to repair and regenerate itself. In addition, the proliferation ability of liver cells is also crucial to the success of liver transplantation surgery, because transplanted liver cells need to be able to proliferate rapidly to adapt to the physiological needs of the recipient. Therefore, the proliferation of liver cells plays an important role in liver health and disease treatment. Summary of the invention

[0004] In order to solve the above problems, the present invention provides N 1 ,N 5 ,N 10 -Application of tri-p-coumaryl spermidine in the preparation of liver drugs.

[0005] This is achieved specifically through the following technical solutions:

[0006] N 1 ,N 5 ,N 10 -Application of tri-p-coumaryl spermidine in the preparation of liver drugs.

[0007] Furthermore, the N 1 ,N 5 ,N 10 -Tri-p-coumaryl spermidine, chemical name N 1 ,N 5 ,N 10 -tri-p-coumaroylspermidine, molecular formula C 34 H 37 N 3 O 6 , molecular weight is 583.7, hereinafter referred to as 3NS, and its specific structural formula is as follows:

[0008]

[0009] Furthermore, the liver medicine is a medicine for preventing and treating liver fibrosis.

[0010] Furthermore, the liver medicine is a medicine for preventing and treating liver damage.

[0011] A liver drug containing N 1 ,N 5 ,N 10 -Tri-p-coumaryl spermidine.

[0012] Furthermore, the drug for treating liver injury includes an oral preparation or an injectable preparation.

[0013] Furthermore, the oral preparation is any one of granules, capsules, tablets, powders, pills, and sustained-release preparations.

[0014] N 1 ,N 5 ,N 10 - Tri-p-coumaryl spermidine can inhibit liver fibrosis by inhibiting the gene expression of fibrosis markers COL1A1, COL1A2, FN1 and CTGF in hepatic stellate cells; it also has a protective effect on liver damage caused by chemicals (including drugs).

[0015] In summary, the beneficial effects of the present invention are: the present invention discloses N for the first time 1 ,N 5 ,N 10 -Tri-p-coumaryl spermidine (chemical name: N 1,N 5 ,N 10 -tri-p-coumaroylspermidine, molecular formula C 34 H 37 N 3 O 6 , molecular weight of 583.7) in the preparation of drugs for the prevention and treatment of liver fibrosis and liver injury. 3NS shows low toxicity and can significantly inhibit the abnormal increase of fibrosis markers COL1A1, COL1A2, FN1 and CTGF in hepatic stellate cells LX-2 cells induced by TGF-β1, while increasing the survival rate of AML-12 liver cells damaged by acetaminophen, and this increase is concentration-dependent, and has a good effect of preventing and treating liver fibrosis and liver injury, and has a certain effect of promoting liver cell proliferation. 3NS not only has a good effect of preventing and treating liver fibrosis, but also has the effect of promoting liver cell proliferation and protecting the liver from liver damage caused by chemical substances, and is therefore suitable for the preparation of drugs for liver-related diseases such as liver fibrosis and liver damage caused by chemical substances or drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the chemical structure of 3NS.

[0017] Figure 2 The figure shows the effect of 3NS at 100 μM on the gene expression levels of fibrosis markers COL1A1, COL1A2, FN1 and CTGF in LX-2 hepatic stellate cells. Compared with the control group, ## P<0.01, ### P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0018] Figure 3 The following is the result of the effect of different concentrations of 3NS on the survival rate of normal AML-12 liver cells. Compared with the control group (CON), # P<0.05, ## P<0.01, ### P<0.001.

[0019] Figure 4 The following is the result of the effect of different concentrations of 3NS on the survival rate of AML-12 hepatocytes damaged by acetaminophen. Compared with the control group, ### P<0.001; compared with the model group, **P<0.01. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments, and any improvement or substitution based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0021] Example 1 demonstrated that 3NS significantly reduced the gene expression of liver fibrosis markers COL1A1, COL1A2, FN1 and CTGF in human hepatic stellate cells LX-2.

[0022] Experimental methods: Real-time fluorescence quantitative PCR was used to detect the effect of 3NS on the mRNA expression of liver fibrosis markers in the TGF-β1-induced LX-2 proliferation model.

[0023] The cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 kU / L penicillin and 100 mg / L streptomycin at 37°C and 5% CO. 2 LX-2 cells were cultured in an incubator. The cell density was adjusted to 17×10 4 / mL, inoculated into 6-well plates, and divided into control group (CON), model group (TGF-β1, 5ng / mL), and drug group (3NS, 100μM). Each group had 3 replicate wells, 2000μL cell suspension in each well, 37℃, 5% CO 2 After culturing for 24 hours under saturated humidity conditions, the drug-treated group was added with the corresponding concentration of drug and cultured without serum for 6 hours. Except for the control group, each well of the other groups was added with culture medium containing TGF-β1 (5 ng / mL) and incubated for 24 hours. The total cell RNA was extracted using the Trizol method, and the RNA concentration and purity were detected using a nucleic acid quantifier. It was immediately reverse transcribed into cDNA. The reverse transcription reaction system and conditions are shown in Tables 1 and 2, and the primer sequences are shown in Table 3. The total volume of the real-time fluorescence quantitative PCR reaction system is 20 μL, including reagents and usage as shown in Table 4. The real-time fluorescence quantitative PCR amplification reaction conditions are: UDG reaction for 2 minutes, 1 cycle; 95°C pre-denaturation for 3 minutes, 1 cycle; cycle reaction at 95°C for 5s, 60°C for 30-34s, and 40 cycles; the melting curve conditions are: instrument default settings. Amplification was performed and 2 -ΔΔCT value.

[0024] Table 1 Reverse transcription reaction system

[0025]

[0026] Table 2 Reverse transcription reaction conditions

[0027]

[0028] Table 3 Primer sequences

[0029]

[0030] Table 4 Real-time fluorescence quantitative PCR reaction system

[0031]

[0032] Experimental Results

[0033] like Figure 2 As shown, the results showed that 100 μM 3NS could significantly reduce the abnormal increase in the levels of cell fibrosis markers COL1A1, COL1A2, FN1 and CTGF induced by TGF-β1. This shows that 3NS has a good anti-liver fibrosis effect and has a good application prospect in the preparation of drugs for the prevention and treatment of liver fibrosis.

[0034] Example 2 demonstrates the safety of 3NS on normal hepatocytes AML-12 and its effect on promoting hepatocyte proliferation

[0035] Experimental method: CCK-8 method was used to detect cell survival rate.

[0036] The cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 kU / L penicillin and 100 mg / L streptomycin, 1% insulin-transferrin-selenium (ITS), and 40 ng / mL dexamethasone (DXMS) at 37°C and 5% CO. 2 AML-12 cells were cultured in an incubator. AML-12 cells in the logarithmic growth phase were taken, digested with 0.25% trypsin, and the cell density was adjusted to 3×10 with culture medium (10% FBS + 1% double antibody + 1% ITS + 40ng / mL DXMS). 4 / mL, inoculated into 96-well plates, and divided into control group (CON) and drug group (6.25, 12.5, 25, 50, 100μM). Each group has 3 replicate wells, 100μL cell suspension in each well, and a blank group with no cells but only culture medium was set up. 37℃, 5% CO 2 After culturing for 24 hours under saturated humidity conditions, add drugs of corresponding concentrations and incubate for 24 hours. Subsequently, add 10 μL of CCK-8 to each well and continue culturing for 1 hour under the same culture conditions. The absorbance (A) at 450 nm of each well was measured by an ELISA instrument, and the cell survival rate was calculated as follows:

[0037]

[0038] Experimental Results

[0039] like Figure 3As shown, the results showed that 3NS had no toxicity to normal AML-12 hepatocytes at a concentration of 6.25-100 μM, and could significantly promote the proliferation of hepatocytes above 25 μM. This showed that 3NS had good safety and could effectively promote the proliferation of hepatocytes, and had a good application prospect in the preparation of drugs for the treatment of liver injury.

[0040] Example 3 demonstrates that 3NS protects normal hepatocytes from acetaminophen-induced damage.

[0041] Experimental method: CCK-8 method was used to detect cell survival rate.

[0042] The culture conditions were the same as those in Experiment 2. The cells were divided into a control group (CON), an APAP (10 mM) group, and a drug group (6.25, 12.5, 25 μM). Each group had 3 replicate wells, and a blank group with no cells but only culture medium was set up. 37°C, 5% CO 2 After culturing for 24 hours under saturated humidity conditions, APAP serum-free culture medium with a final concentration of 10mM was added to each well of the other groups except the control group for incubation for 24 hours, and drugs of corresponding concentrations were added to the drug-treated group for co-incubation. Subsequently, 10μL CCK-8 was added to each well, and the cells were cultured for another 1 hour under the same culture conditions. The absorbance (A) at 450nm of each well was measured with an ELISA instrument, and the cell survival rate was calculated as follows:

[0043]

[0044] Experimental Results

[0045] like Figure 4 As shown in the results, 3NS can significantly improve the survival rate of AML-12 cells induced by acetaminophen in a concentration-dependent manner, indicating that 3NS has a good liver-protective effect and has a good application prospect in the preparation of drugs for the prevention and treatment of liver injury.

[0046] In summary, the present invention provides for the first time an application of 3NS in the preparation of drugs for preventing and treating liver fibrosis and liver injury.

[0047] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from all points of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the equivalent meaning and scope of the claims be included within the scope of protection of the present invention.

Claims

1. N 1 ,N 5 ,N 10 -Application of tri-p-coumaryl spermidine in the preparation of liver drugs.

2. The N according to claim 1 1 ,N 5 ,N 10 - Application of tri-p-coumaryl spermidine in the preparation of liver medicine, characterized in that, The N 1 ,N 5 ,N 10 -Tri-p-coumaryl spermidine, chemical name is N 1 ,N 5 ,N 10 -tri-p-coumaroylspermidine, molecular formula C 34 H 37 N3O6, molecular weight is 583.

7.

3. The N according to claim 1 1 ,N 5 ,N 10 - Application of tri-p-coumaryl spermidine in the preparation of liver medicine, characterized in that, The N 1 ,N 5 ,N 10 -Three pairs of coumaroyl spermidines, the specific structural formula of which is as follows:

4. The N according to claim 1 1 ,N 5 ,N 10 - Application of tri-p-coumaryl spermidine in the preparation of liver medicine, characterized in that, The liver medicine is a medicine for preventing and treating liver fibrosis.

5. The N according to claim 1 1 ,N 5 ,N 10 - Application of tri-p-coumaryl spermidine in the preparation of liver medicine, characterized in that, The liver medicine is a medicine for preventing and treating liver damage.

6. A liver medicine, characterized in that: Contains N 1 ,N 5 ,N 10 -Tri-p-coumaryl spermidine.

7. The liver medicine according to claim 6, characterized in that The medicine for treating liver injury includes oral preparations or injection preparations.

8. The liver medicine according to claim 7, characterized in that The oral preparation is any one of granules, capsules, tablets, powders, pills, and sustained-release preparations.

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