The hepatitis B virus (HBV) is a hepatotropic DNA virus that can cause hepatitis B. If lacking timely and effective treatment, HBV infection can lead to serious consequences such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, which may even endanger the patient's life. Although vaccines can effectively prevent HBV infection, they are ineffective for patients who have already been infected with HBV. Currently, the main anti-HBV drugs used clinically are interferons (IFN) and nucleoside/nucleotide analogues, which function by regulating the immune system and interfering with the replication process of hepatitis B virus, respectively. However, nucleoside/nucleotide analogues can cause drug resistance, and interferons have certain side effects. There is still a lack of effective drugs that can cure HBV infection in clinical practice.

Natural compounds, due to their structural diversity and complexity, are important sources for discovering new drugs or new drug lead molecules. Traditional Chinese medicine records the use of some herbal medicines in the treatment of hepatitis B. In modern pharmaceutical research, many studies have also reported that natural compounds possess inhibitory activity against HBV. Currently, many natural molecules with anti-HBV activity have been discovered, such as terpenoids, lignans, alkaloids, phenolic acids, and flavonoids. Their mechanisms of action mainly include targeting HBV's own life cycle and host cell-related proteins.

1. Exerting Anti-HBV Activity by Regulating HBV's Own Life Cycle

Research has found that asiaticoside, a compound discovered in Hydrocotyle sibthorpioides, can significantly inhibit the formation of HBsAg, HBeAg, HBV DNA, and cccDNA in a dose-dependent manner. Initial exploration of its mechanism of action has revealed that asiaticoside can affect the transcription of corresponding genes and viral replication by inhibiting the activities of the C, S, and X gene promoters.

The caudatin compound isolated from Cynanchum auriculatum has the activity of inhibiting HBsAg secretion and HBV DNA replication. Based on caudatin, a series of new compounds were synthesized through chemical modification, and it was found that 3-O-(3,4,5-trimethoxy) cinnamoyl caudatin exhibited significant inhibitory activity against the secretion of HBsAg, HBeAg, and HBV DNA replication. Initial mechanism exploration revealed that 3-O-(3,4,5-trimethoxy) cinnamoyl caudatin exerts its antiviral effect by interfering with the HBV X promoter and enhancer I, thereby affecting the transcription of the X gene.

HE-145 (helioxanthin) and its analogs isolated from Taiwania cryptomerioides have been found to reduce the levels of HBV DNA and RNA, as well as decrease the expression of core protein. Further exploration of the anti-HBV mechanism of HE-145 revealed that it selectively inhibits the S gene promoter II (SPII) and C gene promoter (CP). However, no inhibition of SPII or CP promoters was observed in non-hepatocytes such as 293T cells and HeLa cells, indicating that the inhibitory effect of HE-145 on SPII and CP is liver-specific. The HBV preC/C promoter/Enh II region contains many transcription factor binding sites, many of which have been shown to play a crucial role in viral gene expression. Electrophoretic mobility shift assay (EMSA) showed that HE-145 reduced the DNA binding activity of several cis-acting elements on CP in HepA2 cell nuclear extracts, including the α-fetoprotein transcription factor binding site, peroxisome proliferators-activated receptors (PPAR) binding site, and transcription factor Sp1 binding site. Overexpression of PPAR and hepatocyte nuclear factor 4 alpha (HNF4α) could alleviate the inhibition of CP by HE-145.

2.Exerting Anti-HBV Activity by Regulating Host Cell-Related Factors

Epigallocatechin-3-gallate (EGCG), a compound found in green tea, can significantly inhibit the secretion of HBsAg and HBeAg in HepG2.2.15 cells in a dose- and time-dependent manner. At the same time, EGCG can also significantly reduce the level of extracellular HBV DNA expression. When Huh7 cells expressing HA-NTCP were treated with EGCG, it led to a decrease in the expression of HA-NTCP protein. Further analysis of the expression of HA-NTCP on the cell membrane revealed that EGCG treatment significantly induced the translocation of NTCP from the cell membrane to the cytoplasm, indicating that EGCG may induce the endocytosis and degradation of NTCP.

Betulinic acid (BetA), extracted from Pulsatilla chinensis, has the effect of inhibiting HBV DNA replication. Animal experiments have found that in hepatocytes derived from HBV transgenic mice, betulinic acid can significantly inhibit HBV replication by downregulating the expression of superoxide dismutase 2 (SOD2), leading to the generation of reactive oxygen species and mitochondrial dysfunction.

The natural phenolic compound curcumin can inhibit the expression and replication of HBV genes. Research has found that this inhibitory effect is mediated by downregulating the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1α is a starvation-induced protein that initiates the gluconeogenesis cascade and has been shown to effectively co-activate HBV transcription. This indicates that curcumin affects PGC-1α at the protein level, inhibiting HBV by promoting the degradation of PGC-1α protein. Additionally, research has also found that curcumin can work synergistically with anti-HBV nucleotide/nucleoside analogues to enhance the inhibition of HBV gene expression. Curcumin, as a host-targeted therapy for HBV infection, can complement current virus-specific treatments.

Oxymatrine (OMTR), derived from the Sophora japonica plant, has been used to treat hepatitis B patients in China for decades, and its safety has been proven. Heat shock cognate protein 70 (Hsc70) is a host protein essential for HBV replication. Research has found that OMTR can significantly downregulate the expression of host Hsc70 mRNA at the post-transcriptional level by disrupting the stability of Hsc70 mRNA, thereby inhibiting the de novo synthesis of HBV from pgRNA to DNA at the reverse transcription stage, thus exhibiting an anti-HBV effect. Since this target is not a viral protein, OMTR is active against both wild-type HBV and strains resistant to reverse transcriptase (RT) inhibitors. In addition, mice with knocked-out Hsc70 genes did not show abnormalities, indicating good safety after inhibiting Hsc70, which also provides a good basis for the further development of OMTR as an anti-HBV drug.

Cepharanthine hydrochloride (CH) is a natural alkaloid derivative. Research has found that this compound can inhibit DNA replication and HBeAg secretion of wild-type or lamivudine-resistant HBV clinical isolates in a dose-dependent manner, suggesting that this compound may be a new alternative drug for treating lamivudine-resistant chronic hepatitis B patients. Further research has revealed that the inhibition of host Hsc70 mRNA expression by CH is parallel to the inhibition of HBV replication within the cell, indicating that CH may inhibit HBV replication by downregulating the expression of host Hsc70.

So far, there are no effective drugs or methods clinically available to cure hepatitis B virus (HBV) infection. Therefore, the research and development of anti-HBV drugs remain a top priority. Based on the current research findings regarding the anti-HBV mechanisms of natural compounds, most of the active compounds inhibit virus replication in cells by acting on host-related factors or regulate virus self-factors and thus affect virus replication by modulating the activities of virus S, C, and X gene promoters. However, there are still very few reports on direct targets of anti-HBV active compounds, and there is a lack of research focused on inhibiting specific stages of the virus's life cycle. Additionally, there has been no discussion of structure-activity relationships or further structural modifications and optimizations for potential anti-HBV natural compounds, making it difficult to provide support for subsequent systematic research on targets and mechanisms. Therefore, as drug researchers, it is essential to conduct in-depth and systematic research on the targets and mechanisms of natural active compounds through interdisciplinary approaches such as chemical biology, to promote the discovery of new lead molecules for natural anti-HBV drugs and new HBV targets.

References

[1] Xiang Jiayao, Xu Min, Feng Yang. Research Progress on the Mechanisms of Natural Compounds Against Hepatitis B Virus (HBV) [J]. Chinese Pharmacological Bulletin, 2021, 37(10): 1346-1351.

About the Author

Xiaomichong, a researcher in pharmaceutical quality, has been dedicated to pharmaceutical quality research and verification of drug analysis methods for a long time. Currently, she works in a large domestic pharmaceutical research and development company, engaged in drug inspection analysis and verification of analytical methods.