Hepatitis B virus (HBV) is one of the most common human pathogens responsible for both acute and chronic hepatitis. Globally, an estimated 257.5 million people were infected with HBV in 2022, with a global HBV prevalence of 3.2%. The global diagnosis and treatment rates for hepatitis B are 14% and 8%, respectively. The global number of hepatitis B related deaths is expected to increase from 858,000 in 2015 to 1.149 million in 2030. During the same period, the number of liver cancer cases is projected to rise from 644,000 to 857,000, and the number of decompensated cirrhosis cases is anticipated to increase from 296,000 to 403,000 (Global prevalence, cascade of care, and prophylaxis coverage of hepatitis B in 2022: A modelling study, 2023). The Diagram of HBV Infection is shown in Figure 1 .

HBV is an enveloped, non-cytolytic virus belonging to the Hepadnaviridae family (Seeger and Mason, 2015). Its genome is a compact, circular, partially double-stranded DNA (3.2 kb) enclosed within a nucleocapsid core and surrounded by an outer lipoprotein layer. The HBV genome contains four overlapping open reading frames (ORFs) (Van Damme et al., 2021). The infection process begins with a low-affinity interaction between viral envelope proteins and heparan sulfate proteoglycan (HSPG), followed by a high-affinity binding to its receptor, sodium taurocholate co-transporting polypeptide (NTCP), which facilitates viral entry (Yan et al., 2012; Verrier et al., 2016). Additionally, the epidermal growth factor receptor (EGFR) facilitates the internalization of HBV into hepatocytes. Once inside the cytoplasm, the virus transports its nucleocapsid into the nucleus, where the viral DNA converts from a relaxed circular form (rcDNA) to covalently closed circular DNA (cccDNA) (Iwamoto et al., 2019; Tsukuda and Watashi, 2020). The cccDNA serves as a template for the production of pregenomic RNA (pgRNA) and various subgenomic RNAs. pgRNA is packaged into the nucleocapsid, where it undergoes reverse-transcription to form the DNA negative strand. The negative strand then serves as a template for the synthesis of the positive strand, resulting in the formation of rcDNA as the major form and dslDNA as the minor form. The rcDNA can be returned to the nucleus and recycled to augment the cccDNA pool. In addition, the nucleocapsid needs to be transported from its location within the cell to the vicinity of the cell membrane. The envelope of HBV consists of three related proteins, called large (L), medium (M), and small (S) hepatitis B surface antigens (HBsAg) (Harvey et al., 1999). The S protein drives the secretion of subviral particles subviral particles (SVPs) (Bi and Tong, 2018). SVPs are particle structures that do not contain the viral genome but play an important role in the interaction between viral infection and the host immune response. Research has shown that SVPs may be a strategic mechanism used by viruses to evade host immune responses (Vaillant, 2021). It has been found that SVPs can interfere with the recognition process of immune cells or induce immune tolerance in immune cells, thus helping the virus escape the attack of the immune system (Selim et al., 2024). L-HBsAg is believed to mediate contact between the virus envelope and nucleocapsid protein (HBcAg) (Tan et al., 1999). In the assembly of infectious virions, the pre-S and S regions of L-HBsAg interact synergistically with hepatitis B core Antigen (HBcAg) (Muhamad et al., 2015).

Currently, the primary therapeutic options for HBV are pegylated-interferon-α (PEG-IFN-α) and nucleos(t)ide analogues (NAs) (Pan et al., 2023). Although these drugs can effectively inhibit HBV replication, reduce inflammation, and improve the prognosis of patients, they are unable to eliminate the initial cccDNA formed in the nuclei of infected liver cells. Consequently, patients typically need to undergo long-term antiviral therapy to suppress viral replication (Martinez et al., 2021; Korkmaz et al., 2023). It is essential to clarify the difference between sterilizing cure and functional cure. A sterilizing cure refers to the complete eradication of HBV, including the elimination of cccDNA from infected hepatocytes, offering the potential for a permanent resolution of the infection. In contrast, a functional cure aims to control the virus to the extent that patients no longer experience disease progression, despite the possible persistence of low levels of cccDNA. Therapeutic strategies targeting cccDNA are crucial for both outcomes. For a functional cure, interfering with cccDNA activity, such as inhibiting its transcription or disrupting its epigenetic regulation, can suppress viral replication to a level where the immune system can maintain control, reducing the risk of cirrhosis and hepatocellular carcinoma. For achieving a sterilizing cure, however, complete elimination of cccDNA is necessary, which requires the development of drugs capable of degrading or removing cccDNA from the nucleus. Therefore, there is an urgent and crucial need to develop effective anti-cccDNA drugs and identify novel targets and methods to disrupt the HBV infection and replication processes, aiming to move closer to either a functional or sterilizing cure for HBV infection.

Literature Search Strategy: A comprehensive literature search was conducted across three authoritative scientific databases: PubMed, Embase, and Web of Science. A set of carefully selected keywords and MeSH terms was utilized to comprehensively cover the research domain. The search terms included “HBV”, “cccDNA”, “Natural products”, “Anti-HBV therapy”, “HBV replication”, “HBV transcription”, “HBx protein”, “HBeAg”, “HBsAg”, “HBc protein”, etc. Boolean operators (AND, OR) were employed to combine these keywords, optimizing the search precision and recall. Our literature search covered all relevant studies from the establishment of the databases to before January 1, 2025.

Inclusion and Exclusion Criteria: Inclusion criteria: Studies were included only if they investigated the effects of natural products on HBV cccDNA, covering aspects such as cccDNA formation, transcription, and stability. Only original research articles based on experimental studies, including cell-based experiments, animal models, or clinical trials, were eligible. Articles had to be published in English. Exclusion criteria: comments, conferences, editorials, letters and replies were excluded. Studies unrelated to HBV cccDNA or natural products were omitted. In cases of duplicate publications, only the article with the most comprehensive.

cccDNA is formed from rcDNA delivered by viral particles and serves as the sole template for viral pgRNA and all subgenomic mRNAs. The conversion of rcDNA to cccDNA involves the following sequential steps: (1) The viral polymerase attached to the 5’-end of the negative strand is removed, resulting in protein-free rcDNA (pf-rcDNA). (2) The RNA primer at the 5’-end of the positive strand is removed. (3) Using the negative strand as a template, the synthesis of the positive strand of viral DNA is completed. (4) The ends of both the positive and negative strands are ligated. (5) The DNA undergoes hyper-helical coiling and associates with histones to form the cccDNA minichromosome ( Figure 1 ) (Zhu et al., 2019). The cccDNA molecule contains four open reading frames (ORFs): surface (S), pre-core/core (C), polymerase (P), and X, which encode seven proteins: HBc (the viral capsid protein), hepatitis B virus polymerase/reverse transcriptase (HBV POL/RT) L, M and S proteins (preS1, preS2 and S domains, which are envelope proteins), and hepatitis B x antigen (HBx, a transcriptional activator). After the pgRNA and polymerase are encapsulated within the nucleocapsids, the pgRNA is reverse transcribed into rcDNA by the viral polymerase (Martinez et al., 2022). Subsequently, a portion of the nucleocapsids are packaged into new infectious particles (Dane particles), while another portion is transported back to the nucleus to replenish the cccDNA pool (Martinez et al., 2022). Consequently, cccDNA is critical for HBV persistence, and even a few copies can sustain infection after treatment cessation (Fanning et al., 2019).

The transcription of cccDNA is a crucial step in the HBV life cycle. This process is regulated by both host factors and viral proteins ( Figure 2 ). Functional cure of hepatitis B is defined as the persistent undetectability of circulating HBsAg and HBV DNA after a finite course of treatment (Wong et al., 2022). Targeting cccDNA transcription is an important means to improve the functional cure rate of hepatitis B. Studies have demonstrated that numerous transcription factors bind to the enhancer or promoter region of the HBV genome, thereby regulating its transcription. These transcription factors include the TATA Box protein (TBP), activating protein 1 (AP-1), specific protein 1 (Sp1), cAMP response element-binding transcription factor (CREB), nuclear respiratory factor 1 (NRF1), and liver tissue-enriched transcription factors such as hepatocyte nuclear factor 1α (HNF1α), HNF3, HNF4α, peroxisome proliferator activated receptor α (PPARα), binding agents (including the two enhancers ENI and ENII), or promoter regions (including the genome containing a core promoter (CP), preS1 promoter (SP1), preS2 promoter (SP2), and X promoter (XP)) (Yoon et al., 2004; Qin et al., 2016; Turton et al., 2020).

Epigenetic modifications of cccDNA, such as DNA methylation and histone modification, play a role in regulating the transcriptional activity of cccDNA. Studies have shown that various host factors including CREB, CREB-binding protein (CBP)/p300, general control nonderepressible 5 (GCN5), Yin Yang 1 (YY1) and CREB-regulated transcriptional coactivator 1 (CRTC1), bind to cccDNA and promote its transcription (Hayashi et al., 2000; Belloni et al., 2009; Tang et al., 2014). Conversely, DNA methyltransferases (DNMT1, DNMT2, DNMT3) cause high methylation of cccDNA by acting on CpG islands in the HBV genome, inhibiting its transcriptional activity (Vivekanandan et al., 2010). In addition, multiple protein methyltransferases, such as protein arginine methyltransferase 1 (PRMT1) and PRMT5, have also been shown to reduce cccDNA transcription by regulating histone methylation levels of cccDNA (Benhenda et al., 2013; Zhang et al., 2017). Histone deacetylases sirtuin 3 (SIRT3), histone deacetylase 1 (HDAC1), HDAC11 reduce histone acetylation levels and inhibit transcriptional activity of cccDNA (Guo et al., 2011; Ren et al., 2018; Yuan et al., 2019). In recent years, with the advancement of high-resolution mass spectrometry, many novel histone modification types, such as crotonylation, succinylation, propionylation and malonylation, have been discovered (Kebede et al., 2017; Wang et al., 2017; Wei et al., 2017; Ishiguro et al., 2018). These findings provide a basis for further understanding of epigenetic modification types of cccDNA.

With the deepening understanding of the role of host factors in the regulation of cccDNA transcription, HBx plays a key role in the initiation and maintenance of cccDNA transcription. Parvulin 14 and Parvulin 17 bind to HBx and cccDNA and promote HBV replication in an HBX-dependent manner (Saeed et al., 2019). When HBx is absent, the histone methyltransferases SETDB1 and PRMT1 bind to cccDNA, causing the transcriptional activity of cccDNA to decrease (Rivière et al., 2015). Studies have shown that when HBx is present, PCAF/GCN5 and CBP/p300 are recruited to cccDNA, and the binding of hSirt1 and HDAC1 to cccDNA is reduced, activating the transcription of cccDNA (Belloni et al., 2009). In addition, HBx also induces host limiting factor Smc5/6, zinc finger E-box binding homeobox 2 (ZEB2), stromal interaction molecule 1 (STIM1) and proteasome activator subunit 4 (PSME4) degradation, enhancing cccDNA transcription activity (Decorsière et al., 2016; Minor et al., 2020). Therefore, HBx itself and HBx-involved protein-protein interactions are novel molecular targets for therapeutic development.

The HBV life cycle exhibits a distinct feature where the genomic DNA, namely rcDNA, is transformed into a molecular template DNA known as cccDNA. This cccDNA serves to amplify a viral RNA intermediate, which is subsequently reverse-transcribed back to viral DNA. The remarkably high stability of cccDNA gives rise to chronic infection and leads to a relatively low cure rate. Therapeutic strategies based on natural products have been found in preclinical trials to be very promising in targeting cccDNA ( Figure 3 ). We reviewed the recently published literature. Natural products play an important role in targeting cccDNA, which is significant for related research and potential therapeutic applications ( Table 1 ).

Natural products encounter significant pharmacokinetic restrictions during their journey towards clinical application for HBV treatment. The most pressing issue is the poor pharmacokinetic properties of natural products, primarily characterized by low solubility and rapid metabolism. For example, curcumin, despite its potent anti-HBV activity in in vitro and some animal models, has limited therapeutic potential due to its hydrophobic nature leading to low oral bioavailability. To overcome this, the development and application of advanced formulation technologies, such as nanocarriers, should be prioritized. Nanocarriers can encapsulate hydrophobic natural products, enhancing their solubility and enabling targeted delivery to liver cells, thereby improving bioavailability.​

Herb-drug interactions also pose significant risks. Given that natural products contain complex mixtures of bioactive compounds that can interact unpredictably with other medications, rigorous clinical trials and in-depth pharmacokinetic studies are essential. These studies should aim to thoroughly understand the interaction mechanisms and establish clear guidelines for the safe co-administration of natural products and conventional drugs.​

The differences between entire extracts and separated substances represent another key challenge. Whole extracts feature multiple components with potential synergistic or antagonistic interactions that affect the overall therapeutic effect, while separated substances may lack certain beneficial properties. Comprehensive comparative studies, integrating chemical analysis with biological activity assays, are needed to determine the most effective form for treatment.​

Batch variability of plant extracts is a major obstacle to standardization and large-scale production. The active components in natural extracts vary greatly depending on factors like the plant’s growth environment, harvest time, extraction methods, and storage conditions. To tackle this, strict quality control measures must be implemented. This includes standardizing the growth and harvest of plant materials, optimizing extraction processes, and establishing comprehensive quality evaluation systems using techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS). By concentrating on these priority areas and advancing along the proposed research directions, substantial progress can be achieved in the clinical utilization of natural products for HBV treatment.

Natural products have emerged as a potential avenue in the fight against HBV infection, particularly in terms of their ability to target cccDNA. HBV has a complex structure. Its virion consists of an outer envelope containing HBsAg. The L protein domain preS1, which binds to NTCP, plays a role in virus entry. It triggers the host immune response and is a major target for diagnostic and therapeutic interventions. The immune response is triggered mainly against the SVPs which are secreted in a high ratio compared to the virions. The functional cure for HBV is defined as undetectable HBsAg, besides undetectable viraemia (Lee and Ahn, 2011). The core of the virus houses the viral genome and is formed by HBc which self-assemble into an icosahedral capsid. Additionally, HBeAg, a secreted protein, is related to viral replication and serves as an indicator of active infection and virus spread (Kao, 2008). The cccDNA, a key reservoir for HBV within hepatocytes, is crucial for the virus’s persistence (Nevola et al., 2023). Several natural compounds, sourced from traditional Thai and Chinese medicines, have demonstrated the ability to reduce cccDNA levels. This reduction is likely associated with their inhibitory effects on viral replication, which may be due to interference with the functions of viral proteins. For instance, the suppression of HBsAg may result from the modulation of viral gene expression or the disruption of viral protein synthesis and trafficking. By targeting cccDNA and related viral processes, these natural products offer the potential for developing novel antiviral therapies to achieve a functional cure for HBV. The following will elaborate on the existing research gaps in several key aspects.

Active component identification and mechanism elucidation: Regarding the application of natural products in anti-HBV research, the relationship between their structures and effects are a crucial aspect. In particular, many natural products are mixtures, which makes the situation even more complicated. Taking the extract of a certain traditional Chinese medicine as an example, it may contain multiple chemical components, such as flavonoids, terpenoids, alkaloids, and so on. These compounds of different structural types may each act on different targets of HBV or enhance the antiviral effect through mutual synergy. For example, flavonoid compounds may inhibit the activity of viral proteins by binding to them, terpenoid compounds may affect the process of viral cell membrane fusion, and alkaloid compounds may interfere with viral gene transcription. However, due to the complexity of the mixtures, accurately identifying the active components and analyzing their structures and action mechanisms face huge challenges. In current research, advanced separation and purification technologies, such as HPLC and MS, need to be used to finely separate and identify the structures of natural product mixtures (Hughes, 2021). Meanwhile, combined with bioactivity tests, the antiviral activities of each component are determined, and then their action mechanisms are further studied.

Efficacy variability and stability: Although natural products have achieved certain results in anti-HBV research, they still face many challenges on the road to clinical application (Atanasov et al., 2021). The efficacy of natural products varies significantly. Taking the traditional solvent extraction method as an example, slight changes in parameters such as the types, concentrations, extraction times, and temperatures of extraction solvents may have a significant impact on the active components in the final extract. In addition, the stability of natural products is also a key factor. Many natural products are prone to undergo chemical reactions such as degradation, oxidation, or isomerization under different environmental conditions, such as light, temperature, and humidity, thereby losing their antiviral activities.

Bioavailability and molecular mechanisms: Poor bioavailability is a common issue with natural products. Many natural compounds have low solubility and are rapidly metabolized, limiting their effectiveness. For example, curcumin exhibits strong anti-HBV activity cell experiments and some animal models. However, due to its poor water solubility, it is difficult to be effectively absorbed by the gastrointestinal tract after oral administration (Mohanty and Sahoo, 2017). The specific molecular mechanism by which natural products act on cccDNA has not been fully elucidated. Although some signaling pathways have been discovered in current research, the details of the precise molecular interactions between these pathways and natural products still need to be further explored. Natural products can interact with multiple intracellular molecules.

Cytotoxicity and quality control: Some natural products may exhibit cytotoxicity at high concentrations. Determining the balance between their anti-HBV activity and cytotoxicity is essential. For natural products that exhibit cytotoxicity at high concentrations, such as C. nutans, the balance between their anti-HBV activity and cytotoxicity needs to be further studied. Such a system includes the use of multiple cell models, a comprehensive assessment of different detection indicators (such as cell viability, apoptosis rate, oxidative stress level, etc.), and a thorough study of the pharmacokinetics and toxicology of drugs in animal models. Natural extracts typically contain multiple active components. The types, contents, and proportions of these components often vary significantly among different source materials and extraction batches. This complexity makes the standardization and quality control of natural products a thorny problem. In terms of quality control, due to the complexity of the components, conventional chemical analysis methods are difficult to comprehensively and accurately detect and monitor the quality of natural products.

Despite the numerous challenges mentioned above, it is encouraging that several classes of highly promising compounds have been identified from a wide range of natural products in existing research. These compounds stand out in terms of their anti-HBV activity, especially in their effects on cccDNA, pointing the way for future research and drug development. The following will provide a detailed introduction to these potentially valuable classes of compounds. Promising Classes of Compounds: Flavonoid-rich compounds like EGCG from green tea show anti-HBV potential, with high-concentration EGCG (22.9μg/ml) reducing cccDNA levels by nearly 60%. Terpenoid-based compounds such as methyl helicterate from Helicteres angustifolia can decrease HBV cccDNA content in vitro and in DHBV-infected ducklings. Alkaloid-containing compounds like DHCH from Corydalis saxicola Bunting can inhibit intracellular HBV cccDNA formation in a dose - and time - dependent manner. These classes of compounds hold promise for anti-HBV drug development.

Future research directions include enhancing the pharmacokinetic properties of natural products. To enhance the pharmacokinetic properties of natural products, we can utilize nanocarrier technology or chemical modification methods. Nanocarriers can encapsulate or adsorb natural products on their surfaces to achieve targeted drug delivery (Solís-Cruz et al., 2021). For example, by designing nanocarriers that specifically target liver cells, the enrichment concentration of natural products in the liver can be increased, their distribution in other tissues can be reduced, thereby reducing potential toxicity and improving antiviral efficacy. Meanwhile, nanocarriers can also protect natural products from the influence of the internal environment of the body and improve their stability (Bennani et al., 2024). Chemical modification can modify the molecular structures of natural products (Ding and Xue, 2024), such as introducing water-soluble groups to improve their solubility or modifying functional groups to enhance their affinity for targets, thereby improving their bioavailability and activity. In-depth exploration of molecular mechanisms should integrate multi-disciplinary methods like genomics, proteomics, and systems biology to study viral genome expression, protein changes, and construct models for predicting drug effects and risks, identifying key targets and biomarkers for personalized medicine. Developing combination therapies by using natural products in conjunction with existing antiviral therapies (such as nucleotide analogues) or other natural compounds can inhibit multiple viral life-cycle stages through different mechanisms, improving efficacy and reducing drug resistance. Additionally, optimizing drug delivery systems for precise liver-targeting and establishing a more comprehensive quality control system with advanced analysis techniques can reduce adverse effects on other tissues and ensure product quality stability, accelerating the translation of natural products from laboratory to clinical applications.

In addition to natural products, emerging non-natural strategies targeting cccDNA are rapidly advancing, offering novel avenues for the complete cure of chronic hepatitis B. The CRISPR-Cas9 system, guided by gRNA, cleaves cccDNA to induce double-strand breaks, leading to sequence mutations or degradation via non-homologous end joining (NHEJ) (Ma et al., 2014). For example, hydrodynamic injection of CRISPR-Cas9 expression vectors enhances cccDNA clearance, though off-target effects and chromosomal translocation risks require attention. Base editing technologies (e.g., cytosine base editors, CBEs) introduce precise point mutations (e.g., C>T) in cccDNA without double-strand breaks, effectively silencing viral gene expression and reducing HBsAg secretion with higher safety profiles (Smekalova et al., 2024). Non-viral delivery systems, such as lipid-like nanoparticles (LLNs) and near-infrared-responsive biomimetic nanoparticles (UCNPs-Cas9@CM), efficiently deliver Cas9/gRNA to the liver, minimizing immunogenicity and off-target rates (Jiang et al., 2017; Wang et al., 2022). Furthermore, small-molecule compounds like disubstituted sulfonamides (DSS) inhibit de novo cccDNA synthesis, while cccDNA reducers (e.g., ccc_R08) selectively reduce cccDNA levels without significant cytotoxicity (Wang et al., 2023). In epigenetic regulation, CRISPR interference (CRISPRi) fuses with KRAB domains to promote heterochromatin formation and silence cccDNA transcription. For instance, siRNA or small molecules targeting HBx (e.g., nitazoxanide) disrupt the HBx-DDB1 complex, restoring SMC5/6-mediated inhibition of cccDNA (Song et al., 2021). These non-natural strategies complement natural products, providing diversified technical pathways to overcome the therapeutic challenge of HBV cccDNA, though further optimization is needed for delivery efficiency, long-term safety, and clinical translation.

In conclusion, natural products have broad application prospects in targeting cccDNA for HBV treatment, but their limitations must be carefully considered before clinical application. Continuous research and development efforts are indispensable to overcome these challenges.