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Application of oleanane triterpenoid compound in resisting hepatitis B virus

A technology of oleanane and triterpenoids, applied in the application field of oleanane triterpenoids in anti-hepatitis B virus

Pending Publication Date: 2020-03-17
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no application of CDDO-related compounds in anti-HBV in the literature

Method used

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  • Application of oleanane triterpenoid compound in resisting hepatitis B virus
  • Application of oleanane triterpenoid compound in resisting hepatitis B virus
  • Application of oleanane triterpenoid compound in resisting hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 The inhibitory effect of oleanane triterpenoids on HBV DNA

[0050] 1. Cell culture

[0051] HepAD38 cell subculture medium (tet+): containing 10% fetal bovine serum (Gbico), 380 μg / ml G418 (Gibco), penicillin and streptomycin double antibody 100 U / ml (Gibco) and 2 μg / mL tetracycline (Tetracycline, tet , Sigma) MEM (Gibco) medium.

[0052] Culture medium (tet-) used for HepGAD38 cell plate / dilution drug: containing 10% fetal bovine serum (Gbico), 380 μg / ml G418 (Gibco), penicillin and streptomycin double antibody 100U / ml (Gibco).

[0053] When the confluence of HepAD38 cells reaches 90%, add 0.25% trypsin-EDTA (Gibco) to the culture flask, digest at 37°C for 5 minutes, discard the trypsin, continue to digest the residual solution at 37°C for 5 minutes, add complete culture solution containing tetracycline Blow off, 1:3 passaging, passaging once every 3-4 days.

[0054] 2. Cytotoxicity detection

[0055] HepAD38 cells were seeded in 96-well plates, 2×10 4 ...

Embodiment 2

[0086] Example 2 The inhibitory effect of oleanane triterpenoids on HBV pgRNA

[0087] HepAD38 cells were seeded in 24-well plates, 1×10 5 Each well was cultured at 37° C. and 5% CO2; after 24 hours, the culture medium in the well plate was discarded, and different concentrations of drugs diluted with complete medium without tetracycline were added. Set up a cell control group (culture medium containing tetracycline and no drug, tet+), a virus control group (culture medium without tetracycline and no drug, tet-) and an experimental drug group (CDDO and CDDO-EA). The same culture medium was replaced once on the 3rd day after adding the drug, and the supernatant liquid was discarded 6 days after the drug was added, and the 24-well plate containing the cells was stored at -80°C for testing.

[0088] 1.1 Total HBV pgRNA in cells

[0089] Intracellular total RNA extraction: add TRIzol (Invitrogen) to 24-well plate, 1 mL / well, lyse on ice for 10 min; add 200 μL chloroform for extr...

Embodiment 3

[0106] Example 3 Effect of oleanane triterpenoids on HBc protein content

[0107] CDDO and CDDO-EA acted on HepAD38 cells with 4 μM 3TC (does not affect HBc and capsid assembly), 2 μM Bay41-4109 (purchased from MedChemExpress Company, reduces HBc content and degrades capsid protein) and 5 μM BA-38017 (Baruch, USA) .Professor Guo Jutao from S Blumberg Institute donated it as a control drug, and the sample collection process was the same as in Example 2.

[0108] Total cell protein lysate preparation (5×): 0.05% bromophenol blue (mass / volume ratio), 0.3M Tris-HCl (pH=6.8), 50% glycerol (volume ratio), 10% SDS (mass / volume ratio ), 25% β-mercaptoethanol (volume ratio).

[0109] Add 100 μL of 1× protein lysate to the 24-well plate cells, lyse on ice for 10 minutes, transfer to EP tube;

[0110] Electrophoresis: prepare stacking gel (concentration is 5%) and separating gel (concentration is 10%), sample carries out SDS-PAGE electrophoresis, adopts voltage 60V in stacking gel, aft...

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Abstract

The invention provides an application of an oleanane triterpenoid compound shown in a formula I, an optical isomer thereof, a solvate or a pharmaceutically acceptable salt thereof in preparation of anti-hepatitis B virus drugs. Wherein R is a hydroxyl group, a C1-10 alkylamino group, a C1-10 alkoxy group, a C1-10 alkyl group, a phenoxy group, a benzyloxy group or an imidazolyl group; the oleananetriterpenoid compound provided by the invention has a remarkable anti-HBV replication effect, and compared with clinically used drugs, the oleanane triterpenoid compound is different in structure andaction mechanism, and has a wide development prospect.

Description

technical field [0001] The invention belongs to the field of medical application, and in particular relates to the application of an oleanane triterpenoid compound in anti-hepatitis B virus. Background technique [0002] Hepatitis B virus (HBV) infection has become a global public health problem. According to the statistics of the World Health Organization in July 2017, there are currently about 257 million HBV infected people in the world. HBV infection can cause acute and chronic hepatitis, which in turn leads to liver lesions. Every year, millions of patients die from liver failure, liver fibrosis, liver cirrhosis and liver cancer caused by HBV infection. my country is a high-incidence area of ​​hepatitis B. Chronic hepatitis B is one of the most common chronic infectious diseases in my country. In 2006, the positive rate of surface antigen reached 7.18%. Although it has decreased to some extent now, it is still around 6%-7%. . [0003] The popularization of HBV vaccine...

Claims

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Application Information

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IPC IPC(8): A61K31/56A61P1/16A61P31/20
CPCA61K31/56A61P1/16A61P31/20
Inventor 李玉环吴硕高强蒋建东
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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